KR20080112359A - Methods for treating conditions associated with masp-2 dependent complement activation - Google Patents

Abstract

In one aspect, the invention provides methods of inhibiting the effects of MASP-2-dependent complement activation in a living subject. The methods comprise the step of administering, to a subject in need thereof, an amount of a MASP-2 inhibitory agent effective to inhibit MASP-2-dependent complement activation. In some embodiments, the MASP-2 inhibitory agent inhibits cellular injury associated with MASP-2-mediated alternative complement pathway activation, while leaving the classical (Clq-dependent) pathway component of the immune system intact. In another aspect, the invention provides compositions for inhibiting the effects of lectin-dependent complement activation, comprising a therapeutically effective amount of a MASP-2 inhibitory agent and a pharmaceutically acceptable carrier. ® KIPO & WIPO 2009

Description

METHODS FOR TREATING CONDITIONS ASSOCIATED WITH MASP-2 DEPENDENT COMPLEMENT ACTIVATION [0002]

The present invention relates to a method of inhibiting the adverse effects of MASP-2-dependent complement activation.

The complement system provides an initial activation mechanism to initiate and amplify inflammatory responses to microbial infections and other acute injuries (MK Liszewski and JP Atkinson, 1993, in Fundamental Immunology, Third Edition, edited by WE Paul, Raven Press, , New York). Although complement activation provides a valuable frontal defense to potential pathogens, activation of complement promoting protective inflammatory responses can be a potential threat to the host (KR Kalli, et al., Springer Semin. Immunopathol. 75: 417-431, 1994, BP Morgan, Eur., J. Clinical Investig., 24: 219-228, 1994). For example, C3 and C5 proteolytic products activate and activate neutrophils. These activated cells release the destructive enzyme indiscriminately and can cause organ damage. Together, complement activation induces adhesion of soluble complement components in the vicinity of the host cell and on the microbial target to dissolve the host cell.

The complement system may be used to treat a number of acute and chronic disease states such as: myocardial infarction, stroke vascular remodeling, ARDS, reperfusion injury, septic shock, thermal burnback capillary leak, inflammation after cardiopulmonary bypass, graft rejection, rheumatoid arthritis , Multiple sclerosis, myasthenia gravis, and the onset mechanism of Alzheimer ' s disease. In most of these symptoms, complement is one of several factors related to the pathogenesis but not the cause. Nonetheless, complement activation may be a key pathological mechanism and may be central to the clinical control of many of these disease states. Increased awareness of the importance of complement-mediated tissue injury in a variety of disease states emphasizes the need for effective complement inhibitors. No drug was administered to humans for the purpose of specifically targeting or inhibiting complement activation.

At present, it is well known that complement systems can be activated through three separate paths: classical pathways, lectin pathways, and alternative pathways. The classical pathway is triggered by antibodies bound to foreign particles (i.e., antigens) and is therefore required prior to exposure to the antigen for the production of specific antibodies. Since activation of the classical pathway is involved in the development of the immune response, the classical pathway is part of the acquired immune system. In contrast, both lectins and alternative pathways are independent of clonal immunity and are part of the innate immune system.

The first step in the activation of the classical pathway is the binding of the specific recognition molecule, C1q, to antigen-binding IgG and IgM. Sequence activation of serine protease enzyme sources is achieved by complement system activation. C1q is a complex of C1, which is bound to the C1r and C1s serine protease proenzyme, and in the binding of C1q to the immune complex, C1r activation of C1s follows self-proteolytic cleavage at the Arg-Ile site of C1r, The resolution of C2 is obtained. C4 is cleaved into two fragments, C4a and C4b, so that the C4b fragment forms a covalent bond with the adjacent hydroxyl or amino group, and the subsequent conversion of the C3 conversion enzyme (C4b2b) through noncovalent interactions with the C2b fragment of activated C2 Generation. The C3 convertase (C4b2b) activates C3, which induces the production of the C5-converting enzyme (C4b2b3b) and the formation of a membrane-attack complex (C5b-9) that can lead to microbial lysis. Activated forms of C3 and C4 (C3b and C4b) are covalently attached on foreign target surfaces recognized by complement receptors on a number of proximal cells.

Independently, the first step in activating the complement system by the lectin pathway is the binding of specific recognition molecules, followed by activation of the putative serine proteases. However, rather than binding of the immune complex by C1q, the recognition molecule in the lectin pathway is carbohydrate-binding proteins (mannan-binding lectin (MBL), H-picoline, M-picoline, and L-picoline) (J. Rev. Immunol. 21: 547-578 (2003); Teh et al., Immunology 101: 225-232 (2000); < RTI ID = 0.0 > Lu et al., Biochim. Biophys. Acta 7572: 387-400,2002; Holmskov et al. )). Ikeda et al., For the first time, demonstrated that, like C1q, MBL can activate the complement system by binding to yeast man-coated red blood cells in a C4-dependent manner (K. Ikeda et al, J. Biol. Chem MBL, a member of the collagen protein family, is a calcium-dependent lectin that binds carbohydrates and 3-and 4-hydroxy groups that are directional to the equatorial plate of the pyranose ring. D-mannose and N-acetyl-D-glucosamine, but carbohydrates unsuitable for steric requirements have an undetectable affinity for MBL (Weis, WI, et al., Nature 360: 127-134, 1992) The interaction between MBL and monosaccharide (Sugar) is typically very weak as a dissociation constant in the range of 2 mM MBL simultaneously achieves specific binding to the glycan ligand by interacting with several monosaccharide residues (Lee, RT, et al., Archiv. Biochem. Biophys. 29 MBL generally recognizes carbohydrate patterns that decorate microorganisms such as bacteria, yeast, parasites and certain viruses, whereas MBL is generally found on mammalian plasma and cell surface glycoproteins Galactose and sialic acid, which decorate the "mature" complex glyco conjugate that binds to the "mature" glycoconjugate, which is known to help protect against self-activation. However, MBL does not recognize the cytoplasm of mammalian cells Linked to a cluster of N-linked glycoproteins isolated on the seam and within the Golgi and high-mannose "precursor" glycans on the glyco lipids (Maynard, Y., et al., J. Biol. Chem. 257: 3788 -3794, 1982). Thus, damaged cells are potential targets of lectin pathway activation through MBL binding.

Picholine processes other types of lectin domains, i.e., fibrinogen-like domains, than MBL. Picoline binds to sugar residues in a Ca < ⁺⁺ > -dependent manner. In humans, three kinds of picoline, L-picoline, M-picoline and H-picoline were identified. The serum picolins, L-picoline and H-picoline, have in common the specificity for N-acetyl-D-glucosamine; However, H-picoline binds to N-acetyl-D-galactosamine. The difference in sugar specificity of L-picoline, H-picoline and MBL means that other lectins can be complementary and, via overlapping, can target other glyco conjugates. This concept is supported by a recent study that, in known lectins within the lectin pathway, only L-picoline alone specifically binds to lipidic tycoic acid, a cell wall glyco conjugate found on all Gram-positive bacteria (Lynch, NJ , et al., J. Immunol. 172: 1198-1202, 2004). Collagen (ie, MBL) and picoline have no significant similarity within the amino acid sequence. However, these two groups of proteins possess a similar domain mechanism and bind to oligomeric structures that maximize the potential for multisite binding, such as C1q. Serum concentrations of MBL are highly variable in healthy populations, which are genetically controlled by gene polymorphisms / mutations in both the promoter and coding domains of the MBL gene. In acute phase proteins, the expression of MBL is further up-regulated in the inflammatory process. L-picoline is present in serum at similar concentrations as MBL. Thus, the L-picoline arm of the lectin pathway is potentially compared to the MBL arm in intensity. MBL and picoline may also function as opsonins, which require the interaction of these proteins with phagocyte receptors (Kuhlman, M., et al, J. Exp. Med. 169: 1733, 1989; Matsushita, M., et al, J. Biol. Chem. 271: 2448-54, 1996). However, the characteristics of the receptor (s) on the phagocytic cells have not yet been established.

Human MBL forms a specific, high-affinity interaction with specific C1r / C1s-like serine proteases through their collagen-like domains, that is, MBL-cancere serine proteases (MASPs). To date, three MASPs have been described. First, a single enzyme "MASP" has been identified and characterized as an enzyme responsible for initiation of the complement cascade (i.e., C2 and C4 cleavage) (Ji, YH, et al., J. Immunol. 150: 571-578, 1993 ). It then proved that MASP is a mixture of two proteases: MASP-1 and MASP-2 (Thiel, S., et al, Nature 386: 506-510, 1997). However, it has been demonstrated that MBL-MASP-2 complex alone is sufficient for complement activation (Vorup-Jensen, T., et al., J. Immunol. 165: 2093-2100, 2000). In addition, only MASP-2 disrupted C2 and C4 at high speed (Ambrus, G., et al., J. Immunol. 170: 1374-1382, 2003). Thus, MASP-2 is a protease that activates C4 and C2 to produce C3 conversion enzyme, C4b2b. This is a significant difference from the C1 complex, where the coordination reaction of two specific serine proteases (C1r and C1s) induces activation of the complement system. Recently, a third novel protease, MASP-3, has been isolated (Dahl, M. R., et al., Immunity 15: 127-35, 2001). MASP-1 and MASP-3 are alternatively spliced products of the same gene. The biological functions of MASP-1 and MASP-3 remain dissolved.

MASPs share the same domain machinery as their C1r and C1s, the enzyme component of the C1 complex (Sim, R.B., et al., Biochem. Soc. Trans. 28: 545, 2000). These domains include the N-terminal Clr / Cls / sea urchin Vegf / Bone forming protein (CUB) domain, epidermal growth factor-like domain, second CUB domain, tandem of the complement control protein domain, and serine protease domain. In C1 protease, activation of MASP-2 occurs through cleavage of the Arg-Ile linkage adjacent to the serine protease domain, which divides the enzyme into disulfide linked A and B chains, the latter consisting of the serine protease domain. Recently, a genetically determined deficiency of MASP-2 has been reported (Stengaard-Pedersen, K., et al., New Eng. J. Med. 349: 554-560, 2003). Mutations of a single nucleotide induce Asp-Gly exchange in the CUBI domain and confer MASP-2 which does not bind to MBL.

MBL is a protein that binds to the mRNA of 19 kDa MBL-related protein (MAp 19) (Stover, CM., J. Immunol. 162: 3481-90, 1999) or a small MBL-associated protein (sMAP) (Takahashi, M., et al. Int. Immunol. 11: 859-863, 1999). MAp19 is formed by an alternative splicing of the MASP2 gene product and contains the first two domains of MASP-2 followed by an additional sequence of four unique amino acids. The MASP 1 and MASP 2 genes are located on chromosome 3 and 1, respectively (Schwaeble, W., et al., Immunobiology 205: 455-466, 2002). Several evidences have demonstrated that there is another MBL-MASP complex and that a large portion of the total MASP in the serum is not associated with MBL (Thiel, S., et al., J. Immunol. 165: 878-887 , 2000). Both H- and L-picolines are associated with MASP and activate the lectin complement pathway as MBL (Dahl, MR, et al., Immunity 15: 127-35, 2001; Matsushita, M., et al. , J. Immunol. 168: 3502-3506, 2002). Both the lectin and the classical pathway form the common C3 convertase (C4b2b), and both pathways are concentrated at this stage.

The lectin pathway is known to play a major role in host defense against infection. Strong evidence for MBL involvement in host defense has been shown in the analysis of patients with reduced serum levels of functional MBL (Kilpatrick, D. C, Biochim. Biophys. Acta 1572: 401-413, 2002). These patients exhibit susceptibility to recurrence of bacterial and fungal infections. These symptoms are evident early in life before development of a full repertoire of antibody responses in the apparent window process of vulnerability by attenuating the maternal inductive antibody titer. This syndrome often causes mutations at some sites within the collagen part of MBL, which interferes with the proper formation of MBL oligomers. However, since MBL functions as a complement independent obsonin, it has not been revealed that the degree of susceptibility to increased infection is due to impaired complement activation.

The role of the lectin pathway has begun to be evaluated, although there is widespread evidence that it is involved in the classical and alternative complement pathway within the pathogenesis of non-infectious human disease. Recent studies provide evidence that activation of the lectin pathway can lead to complement activation and inflammation in ischemia / reperfusion injury. Collard et al. (2000) reported that cultured endothelial cells with oxidative stress adhere to MBL and show C3 attachment by exposure to human serum (Collard, CD, et al., Am. J. Pathol. : 1549-1556, 2000). In addition, treatment of human serum with blocking anti-MBL mAb inhibited MBL binding and complement activation. These results extend to the RET model of myocardial ischemia-reperfusion, and it appears that irete treated with blocking antibodies to RET MBL significantly reduces myocardial damage due to cardiac artery occlusion than does the RET treated with control antibody (Jordan, JE, et al., Circulation 104: 1413-1418, 2001). The molecular mechanism of MBL binding to vascular endothelium after oxidative stress is unclear; Recent studies have suggested that activation of the lectin pathway after oxidative stress may be mediated by MBL bound to vascular endothelial cytokeratin rather than glyco conjugate (Collard, CD, et al., Am. J. Pathol. 159: 1045-1054, 2001). Other studies have focused on classical and alternative pathways in the pathogenesis of ischemia / reperfusion injury, and the role of the lectin pathway in this disease is controversial (Riedermann, NC, et al., Am. J. Pathol. -367, 2003).

In contrast to the classical and lectin pathway, no initiator of the alternative pathway has been found to perform the recognition function that C1q and the lectin perform in the other two pathways. It is widely accepted that it is now spontaneously triggered by foreign or other abnormal surfaces (bacteria, yeast, virus infected cells, or damaged tissue). There are four plasma proteins directly involved in the alternative pathway: C3, Factors B and D, and propers. The proteolytic production of C3b to C3 requires an alternative pathway to function. Since the alternative pathway C3 converting enzyme (C3bBb) contains C3b as an essential subunit, the question of the origin of the first C3b through the alternative path has presented a complicated problem and has stimulated considerable research.

C3 belongs to a family of proteins (C4 and alpha-2 macroglobulin) that contains a rare post-translational modification known as a thioester linkage. The thioester group is composed of glutamine in which the terminal carbonyl group is bonded to the sulfhydryl group of the cysteine 3 amino acid. This bond is unstable, and the electrophilic carbonyl group of glutamine can form a covalent bond with another molecule through a hydroxyl or amino group. The thioester bond is quite stable when isolated within the hydrophobic pocket of intact C3. However, C3 is proteolytically cleaved into C3a and C3b and is exposed to a highly reactive thioester bond on C3b, by which C3b is covalently attached to the target. In addition to the well-documented role of C3b covalently binding to complement targets, C3 thioesters play a major role in triggering alternative pathways. In accordance with, an alternate path is a fluid-well "over theory tick" is received the switch enzyme, is initiated by the generation of iC3Bb, hydrolyzed thioester to _{C3 (iC3; C3 (H 2} O)) and factor B (Lachmann, PJ, et al., Springer Semin. Immunopathol. 7: 143-162, 1984). C3b-like iC3 is fished from native C3 by low-speed spontaneous hydrolysis of internal thioester in the protein (Pangburn, MK, et al., J. Exp. Med. 754: 856-867, 1981). Through activation of the iC3Bb converting enzyme, the C3b molecule is attached on the target surface, thus initiating the alternative pathway.

There is little known about the initiator of alternate path activation. Activators are known to include yeast cell walls (zymosan), many pure polysaccharides, levitic erythrocytes, specific immunoglobulins, viruses, fungi, bacteria, animal tumor cells, parasites, and damaged cells. The only common feature of these activators is the presence of carbohydrates, and the complexity and diversity of the carbohydrate structure makes it difficult to form a recognized covalent molecular determinant.

The alternative pathway provides a robust amplification loop of lectin / classical pathway C3 converting enzyme (C4b2b) because factor B is involved in all C3b generated in the formation of an additional alternative pathway C3 converting enzyme (C3bBb). The alternative pathway C3 converting enzyme is stabilized by binding of propers. Proprin enhances the half-life of alternative pathway C3 converting enzyme by 6 to 10 fold. C3 < / RTI > converting enzyme by inducing the formation of an alternative pathway C5 converting enzyme.

All three pathways (i.e., classical, lectin and alternative pathways) are known to be concentrated in C5, which is cleaved to form a product with a number of pro-inflammatory effects. The concentrated path is called the end complement path. C5a is the most potent anapylatoxin that induces smooth muscle and vascular tone, and changes in vascular permeability. It is the most potent chemotactic and activator of both neutrophils and monocytes. C5a-mediated cellular activation can significantly increase the inflammatory response by inducing the release of a number of additional inflammatory mediators, including cytokines, hydrolases, arachidonic acid metabolites and reactive oxygen species. C5 cleavage leads to the formation of C5b-9, also known as the membrane attack complex (MAC). There is strong evidence that low-soluble MAC attachment plays an important role in inflammation as well as its role as a soluble pore-forming complex.

Summary of the Invention

As one aspect of the present invention, the present invention provides a method for inhibiting the adverse effects of MASP-2-dependent complement activation in a living subject. The method comprises administering to a subject in need thereof an amount of a MASP-2 inhibitor effective to inhibit MASP-2-dependent complement activation. As used herein, the expression "MASP-2-dependent complement activation" refers to alternative pathway complement activation that occurs through a lectin-dependent MASP-2 system. In another aspect of the invention, the MASP-2 inhibitor inhibits complement activation through a lectin-dependent MASP-2 system, without substantially inhibiting complement activation through a classical or C1q-dependent system such that the C1q- .

In one embodiment of this aspect of the invention, the MASP-2 inhibitor is an anti-MAST-2 antibody or a fragment thereof. In another embodiment of the invention, the anti-MASP-2 antibody possesses a reduced effector function. In one embodiment, the MASP-2 inhibitor is a MASP-2 inhibitory peptide or a non-peptide MASP-2 inhibitor.

In another aspect, the invention provides a composition for inhibiting the adverse effects of MASP-2-dependent complement activation comprising a therapeutically effective amount of a MASP-2 inhibitor and a pharmaceutically acceptable carrier. The methods comprise a therapeutically effective amount of a MASP-2 inhibitor in a pharmaceutical carrier and provide a method for the manufacture of a medicament for use in inhibiting the adverse effects of MASP-2-dependent complement activation in a living subject in need thereof. The method provides for the manufacture of a medicament for use in inhibiting MASP-2-dependent complement activation for the treatment of each of the conditions, diseases and disorders described below.

The methods, compositions and medicaments of the present invention are useful for inhibiting the in vivo adverse effects of MASP-2-dependent complement activation in mammalian subjects, including humans, suffering from acute or chronic pathological conditions or damage as described further herein. Such symptoms and impairments include, but are not limited to, MASP-2 mediated complement activation in autoimmune diseases and / or inflammatory conditions.

As a feature of the present invention, there is provided a method of treating aortic aneurysm repair, cardiopulmonary bypass surgery such as organ transplantation (e.g., heart, lung, liver, kidney) and / or limb / fingers remodeling, stroke, myocardial infarction, Methods for treating ischemic reperfusion injury by treating a subject suffering from ischemic reperfusion, including but not limited to vascular relapses related to hemodynamic resuscitation and / or surgical operations, with a therapeutically effective amount of a MASP-2 inhibitor in a pharmaceutical carrier .

As a feature of the present invention, there is provided a method of inhibiting atherosclerosis by treating a subject suffering from or susceptible to atherosclerosis with a therapeutically effective amount of a MASP-2 inhibitor in a pharmaceutical carrier.

In one aspect of the invention, there is provided a method of treating a patient suffering from cardiovascular, cerebrovascular, peripheral (e.g., musculoskeletal) vascular conditions, renal vascular symptoms, mesenteric / Dependent complement activation in a subject that experiences vascular symptoms including, but not limited to, vascular remodeling in re-transplantation. These symptoms include: vasculitis, such as Henoch-Schlenz spinal nephritis, systemic lupus erythematosus-vasculitis, vasculitis associated with rheumatoid arthritis (so-called malignant rheumatoid arthritis), immune complex vasculitis, and Takayama sickness; Dilated cardiomyopathy; Diabetic angiopathy; Kawasaki disease (arteritis); Venous air embolism (VGE); And / or post-stent restenosis, rotator cuff resection, and / or percutaneous transluminal coronary angioplasty (PTCA).

As another feature of the present invention there is provided a method of inhibiting MASP-2-dependent complement activation in a subject suffering from inflammatory gastrointestinal disorders including, but not limited to, pancreatitis, diverticulitis and bowel disorders such as Crohn's disease, ulcerative colitis, and irritable bowel syndrome ≪ / RTI >

Another aspect of the present invention is to provide a method of treating acute respiratory distress syndrome, transfusion-related acute lung injury, ischemia / reperfusion acute lung injury, chronic obstructive pulmonary disease, asthma, Wegener's granulomatosis, In a subject suffering from pulmonary circulation including, but not limited to, pulmonary fibrosis, pulmonary fibrosis, pulmonary bronchitis syndrome, idiopathic pulmonary fibrosis, secondary acute lung injury due to burn, non-cardiogenic pulmonary edema, transfusion-related respiratory depression, Dependent < / RTI >

Other features of the present invention include, but are not limited to, hemodialysis, plasma exchange, leukapheresis, ECMO, heparin-induced maximal oxygenated LDL deposition (HELP) Dependent complement activation in a subject who has, is, or will undergo an unrestricted extracorporeal reperfusion process.

Other aspects of the invention include, but are not limited to, osteoarthritis, rheumatoid arthritis, rheumatoid arthritis, gout, neuropathic arthropathy, psoriatic arthritis, ankylosing spondylitis or other vertebral arthritis and crystalline arthropathies, muscle regressive atrophy or systemic lupus erythematosus Dependent complement activation in a subject suffering from a musculoskeletal condition that is not limited to the MASP-2-dependent complement.

Other characteristics of the present invention include glomerulonephritic glomerulonephritis, glomerulonephritis, proliferative glomerulonephritis (glomerular capillary glomerulonephritis), glomerulonephritis after acute infection (glomerulonephritis after infection with streptococci), cold glomerulonephritis, Dependent complement activation in a subject suffering from kidney conditions including, but not limited to, Lupus nephritis, Henoch-Shenline spinal nephritis or IgA nephropathy.

As another aspect of the present invention, there is provided a method of treating a patient suffering from psoriasis, autoimmune spastic dermatosis, eosinophil-enhanced cotton, bullous pemphigoid pheochromocytoma, acquired vesicular epidermolysis and gestational herpes and other skin disorders, or capillary leak due to thermal or chemical burn injury Dependent complement activation in a subject suffering from, but not limited to, skin conditions.

As another feature of the present invention, there is provided a method for the treatment or prevention of a cancer, including transplantation or heterologous transplantation of whole organs (e.g., kidney, heart, liver, pancreas, lung, cornea, etc.) or graft Dependent complement activation in a subject having an unrestricted organ or other tissue transplantation.

Another aspect of the present invention is a method of treating multiple sclerosis (MS), myasthenia gravis (MG), Huntington's chorea (HD), amyotrophic lateral sclerosis (ALS), acute idiopathic polyneuritis, post-stroke reperfusion, In a subject suffering from a central nervous system disorder or impairment or peripheral nervous system disorder or impairment including, but not limited to, Parkinson's disease (PD), Alzheimer's disease (AD), Miller-Fisher syndrome, brain trauma and / Lt; RTI ID = 0.0 > MASP-2-dependent < / RTI >

As a further feature of the present invention there is provided a method of treating a subject suffering from a blood disorder including but not limited to severe sepsis, septic shock, acute respiratory distress syndrome due to sepsis, and consequences of sepsis or sepsis including without limitation systemic inflammatory response syndrome Lt; RTI ID = 0.0 > MASP-2-dependent < / RTI > Related methods provide methods for treating other blood disorders including bleeding shock, hemolytic anemia, autoimmune thrombotic hemolytic anemia (TTP), hemolytic uremic syndrome (HUS), or other bone marrow / blood breakdown symptoms.

As another feature of the present invention, there is provided a method of treating a patient suffering from a genitourinary symptom including but not limited to pain bladder disease, sensory bladder disease, chronic aseptic cystitis and interstitial cystitis, male and female infertility, placental dysfunction and lactation, Lt; RTI ID = 0.0 > MASP-2-dependent < / RTI >

As another feature of the present invention there is provided a method of treating a patient suffering from vascular disease, neuropathy or retinopathy complications of non-BMI diabetes (type 1 diabetes or insulin dependent diabetes) or type 1 or type 2 (adult type) 2-dependent < / RTI > complement activation.

In another aspect of the present invention there is provided a method of treating a patient in need of such treatment comprising administering to the patient a MASP-2 inhibitor before or after, before and / or after administration of chemotherapeutic agent (s) and / or radiation therapy 2-dependent complement activation in a subject to be treated with a chemotherapeutic agent and / or radiotherapy that includes, without limitation, the treatment of cancerous conditions by administering a therapeutically effective amount of MASP-2. The administration of post-chemotherapy or post-war radiation therapy of MASP-2 inhibitors is useful for reducing the side effects of chemotherapeutic agents or radiotherapy. As a further feature of the present invention, there is provided a method of treating malignant tumors by administering a MASP-2 inhibitor in a pharmaceutically acceptable carrier to a malignant tumor patient.

Another aspect of the invention provides a method of inhibiting MASP-2-dependent complement activation in a subject suffering from endocrine disorders by administering to such a subject a therapeutically effective amount of a MASP-2 inhibitor in the pharmaceutical carrier. Symptoms of treatment according to the present invention include, but are not limited to, Hashimoto's thyroiditis, stress, anxiety and other potential hormonal disorders associated with prolactin, growth or insulin-like growth factors from the pituitary gland and controlled release of adrenocorticotropin .

As another feature of the present invention, there is provided a method of treating a subject suffering from age-related macular degeneration or other complement mediated scientific symptoms by administering to a subject having such symptoms a therapeutically effective amount of a MASP-2 inhibitor in a pharmaceutical carrier, Thereby providing a method of inhibiting complement activation.

BRIEF DESCRIPTION OF THE DRAWINGS The foregoing features and many of the advantages of the invention will be more clearly understood from the following detailed description taken in conjunction with the accompanying drawings, in which:

Figure 1 is a flow chart showing a new discovery that an alternative complement pathway requires lectin pathway-dependent MASP-2 activation for complement activation;

Figure 2 is a diagram showing the genomic structure of human MASP-2;

3A is a diagram showing the domain structure of a human MASP-2 protein;

Figure 3B is a diagram showing the domain structure of the human M Ap19 protein;

4 is a diagram illustrating a murine MASP-2 knockout strategy;

Figure 5 is a representation of a human MASP-2 minigene construct;

Figure 6A provides results demonstrating that MASP-2 deficiency induces loss of lectin-pathway mediated C4 activation as measured by lack of C4b attachment on the metaphase;

Figure 6B provides results demonstrating that MASP-2 deficiency induces lectin-pathway mediated C4 activation loss as measured by C4b attachment deficiency on gemic acid;

Figure 6C shows MASP-2 +/- measured by C4b attachment of mannan and gemic acid; MASP-2 - / - and wild-type strains;

7A provides results demonstrating that MASP-2 deficiency induces loss of both lectin-pathway mediated and alternative pathway mediated C3 activation measured by C3b adhesion deficiency on metastasis;

Figure 7B provides results demonstrating that MASP-2 deficiency induces loss of both lectin-pathway mediated and alternative pathway mediated C3 activation as measured by C3b adhesion deficiency on the gemic acid phase;

Figure 8 provides results demonstrating that murine recombinant MASP-2 restores lectin-pathway-mediated C4 activation in a protein concentration-dependent manner measured by C4b attachment of mastin phase with the addition of MASP-2 - / - serum samples do;

Figure 9 provides results demonstrating that the classical pathway is functional within the MASP-2 - / - strain;

Figure 10 provides results demonstrating that the MASP-2-dependent complement activation system is activated in the ischemia / reperfusion phase following abdominal aortic aneurysm repair;

11A provides results demonstrating that anti-MASP-2 Fab2 antibody # 11 inhibits C3 convertase formation as described in Example 24;

Figure 11B provides results demonstrating that anti-MASP-2 Fab2 antibody # 11 binds to native retest MASP-2 as described in Example 24;

Figure 11C provides results demonstrating that anti-MASP-2 Fab2 antibody # 41 inhibits C4 cleavage, as described in Example 24;

Figure 12 provides results demonstrating that all anti-MASP-2 Fab2 antibodies, as described in Example 24, were tested for inhibition of C4 cleavage by inhibited C3 converting enzyme formation;

Figure 13 is a representation of a recombinant polypeptide derived from rat MASP-2 used for epitope mapping of anti-MAASP-2 blocking Fab2 antibodies as described in Example 25;

Figure 14 provides results demonstrating that anti-MASP-2 Fab2 # 40 and # 60 bind to rat MASP-2 polypeptide as described in Example 25;

Figure 15 demonstrates the removal of blood urea nitrogen from wild type (+ / +) and MASP-2 (- / -) mice at 24 and 48 hours after reperfusion in a renal ischemia / reperfusion injury model as described in Example 26 Provide results;

Figure 16A provides results demonstrating the infarct size of post-injury wild type (+ / +) and the reduced infarct size of MASP-2 (- / -) mice in a coronary artery occlusion and reperfusion model as described in Example 27 do;

Figure 16B provides results showing the classification of individual animals tested in a coronary artery occlusion and perfusion model as described in Example 27;

Figure 17A provides results showing basal VEGF protein levels in the RPE-choroid complex isolated in wild-type (+ / +) and MASP-2 (- / -) mice as described in Example 28;

Figure 17B shows the levels of VEGF protein in RPE-choroid complexes induced in wild type (+ / +) and MASP-2 (- / -) mice at 3 days after laser induced injury in the macular degeneration model as described in Example 28 Provide the result that it represents;

Figure 18 shows the results of mean colloid neovascularization (CNV) volume at 7 days following laser induced injury in wild type (+ / +) and MASP-2 (- / -) mice as described in Example 28 to provide;

Figure 19 provides results showing the mean clinical arthritis score of wild type (+ / +) and MASP-2 (- / -) mice after Col2 mAb-induced rheumatoid arthritis as described in Example 29;

20A is a diagram showing the targeted destruction of the sMAP (Map 19) gene as described in Example 30;

Figure 2OB provides Southern blot analysis of genomic DNA isolated from progeny induced by mating of male sMAP (- / -) chimeric mice and female C57BL / 6 mice as described in Example 30;

Figure 2OC provides PCR genotypic analysis of wild type (+ / +) and sMAP (- / -) mice as described in Example 30;

21A provides Northern blot analysis of sMAP and MASP-2 mRNA in sMAP (- / -) mice as described in Example 30;

Figure 21B shows the quantitative RT-PCR of cDNA encoding MASP-2 H-chain, MASP-2 L-chain and sMAP in wild type (+ / +) and sMAP (- / - PCR analysis;

Figure 22A provides an immunoblot demonstrating a deficiency of sMAP (- / -), i.e., MAp19 (- / -), MASP-2 and sMAP in mouse serum, as described in Example 30;

Figure 22B provides results demonstrating that MASP-2 and sMAP were detected in the MBL-MASP-sMAP complex as described in Example 30;

Figure 23A provides results showing C4 attachment on mannan-coated wells in wild-type (+ / +) and sMAP (- / -) mouse sera as described in Example 30;

Figure 23B provides results showing C3 attachment on mannan-coat wells in wild-type (+ / +) and sMAP (- / -) mouse sera as described in Example 30;

24A provides results showing reconstitution of the MBL-MASP-sMAP complex in sMAP (- / -) serum as described in Example 30;

Figures 24B-D provide results showing that rsMAP and MASP-2i competitively bind to MBL as described in Example 30;

25A-B provide results showing storage of C4 adherence activity by addition of rMASP-2 but not rsMAP as described in Example 30;

Figures 26A-B provide results showing the decrease in C4 adherence activity by addition of rsMAP as described in Example 30; And

Figures 27A-C provide results showing that MASP-2 is responsible for C4 bypassing activation of C3, as described in Example 31.

Explanation of sequence list

SEQ ID NO: 1 human MAp 19 cDNA

SEQ ID NO: 2 human MAp 19 protein (including leader)

SEQ ID NO: 3 human MApl9 protein (maturation)

SEQ ID NO: 4 human MASP-2 cDNA

SEQ ID NO: 5 human MASP-2 protein (including leader)

SEQ ID NO: 6 human MASP-2 protein (maturation)

SEQ ID NO: 7 human MASP-2 gDNA (exons 1-6)

Antigen: (see MASP-2 mature protein)

SEQ ID NO: 8 CUBI sequence (aa 1-121)

SEQ ID NO: 9 CUBEGF sequence (aa 1-166)

SEQ ID NO: 10 CUBEGFCUBII (aa 1-293)

SEQ ID NO: 11 EGF region (aa 122-166)

SEQ ID NO: 12 serine protease domain (aa 429-671)

SEQ ID NO: 13 Serine protease domain inactivation (aa 610-625, including Ala mutation of Ser618)

SEQ ID NO: 14 TPLGPKWPEPVFGRL (CUBI peptide)

SEQ ID NO: 15

TAPPGYRLRLYFTHFDLELSHLCEYDFVKLSSGAKVLATLC

GQ (CUBI peptide)

SEQ ID NO: 16 TFRSDYSN (MBL binding domain core)

SEQ ID NO: 17 FYSLGSSLDITFRSDYSNEKPFTGF (MBL binding region)

SEQ ID NO: 18 IDECQVAPG (EGF peptide)

SEQ ID NO: 19 ANMLCAGLESGGKDSCRGDSGGALV (serine protease binding core)

Peptide inhibitors:

SEQ ID NO: 20 MBL full length cDNA

SEQ ID NO: 21 MBL full length protein

SEQ ID NO: 22 OGK-X-GP (consensus binding)

SEQ ID NO: 23 OGKLG

SEQ ID NO: 24 GLR GLQ GPO GKL GPO G

SEQ ID NO: 25 GPO GPO GLO GLQ GPO GKL GPO GPO GPO

SEQ ID NO: 26 GKDGRDGTKGEKGEPGQGLRGLQGPOGKLGPOG

SEQ ID NO: 27 GAOGSOGEKGAOGPQGPOGPOGKMGPKGEOGDO (human h-picoline)

SEQ ID NO: 28

GCOGLOGAOGDKGEAGTNGKRGERGPOGPOGKAGPOGPN

GAOGEO (human picoline p35)

SEQ ID NO: 29 LQRALEILPNRVTIKANRPFLVFI (C4 cleavage site)

Expression inhibitors:

The cDNA of SEQ ID NO: 30 CUBI-EGF domain (nucleotide of SEQ ID NO: 4

22-680)

SEQ ID NO: 31

5 'CGGGCACACCATGAGGCTGCTGACCCTCCTGGGC 3'

The nucleotide sequence of SEQ ID NO: 4 containing the MASP-2 translation initiation site (sense)

12-45

SEQ ID NO: 32

5 'GACATTACCTTCCGCTCCGACTCCAACGAGAAG 3'

 MASP-2 < / RTI > MBL binding site (sense)

Of nucleotides 361-396

SEQ ID NO: 33

5 'AGCAGCCCTGAATACCCACGGCCGTATCCCAAA 3'

 The nucleotide sequence of SEQ ID NO: 4 encoding the region comprising the CUBII domain

Tide 610-642

Cloning primer:

SEQ ID NO: 34 CGGGATCCATGAGGCTGCTGACCCTC (5 'PCR for CUB)

SEQ ID NO: 35 GGAATTCCTAGGCTGCATA (3 'PCR for CUB)

SEQ ID NO: 36 GGAATTCCTACAGGGCGCT (3 'PCR for CUBIEGF)

SEQ ID NO: 37 GGAATTCCTAGTAGTGGAT (3 'PCR for CUBIEGFCUBII)

SEQ ID NOS: 38-47 is a cloning primer for human adaptive antibodies

SEQ ID NO: 48 is a 9 aa peptide bond

Expression vector:

SEQ ID NO: 49 is a MASP-2 mini gene insert

SEQ ID NO: 50 is a murine MASP-2 cDNA

SEQ ID NO: 51 is a murine MASP-2 protein (w / leader)

SEQ ID NO: 52 is a mature murine MASP-2 protein

SEQ ID NO: 53 Rett MASP-2 cDNA

SEQ ID NO: 54 is the ret MASP-2 protein (w / leader)

SEQ ID NO: 55 is a mature rat MASP-2 protein

SEQ ID NO: 56-59 is a site-directed mutagenic oligonucleotide of human MASP-2 used to generate human MASP-2A

SEQ ID NO: 60-63 is a site-directed mutagenic oligonucleotide of murine MASP-2 used to generate murine MASP-2A

SEQ ID NO: 64-65 is a site-directed mutagenic oligonucleotide of ret MASP-2 used to generate rat MASP-2A

DETAILED DESCRIPTION OF THE INVENTION

The present invention is based on the discovery of the surprising fact that MASP-2 is required for the initiation of alternative complement pathway activation. Using the MASP-2 - / - knockout mouse model, we have shown that it is possible to inhibit alternative complement pathway activation through the lectin-mediated MASP-2 pathway without compromising the classical pathway, and in the absence of the classical pathway Lt; RTI ID = 0.0 > MASP-2 < / RTI > activation as a requirement for alternative complement activation. The present invention also discloses the use of MASP-2 as a therapeutic target for inhibiting cellular damage associated with lectin-mediated alternative complement pathway activation without compromising the classical (C1q-dependent) pathway component of the immune system.

I. Definition

Unless defined otherwise herein, all terms herein have the same meaning as understood by those skilled in the art. The following definitions are provided to provide clarity as to terms used in the specification and claims of the invention.

As used herein, the term "MASP-2-dependent complement activation" is an alternative pathway complement activation that occurs through lectin-dependent MASP-2 activation.

As used herein, the term "alternative pathway" is intended to include, but is not limited to, for example, zymosan, gram negative membranes and levitic erythrocytes of fungal and yeast cell walls, and a number of pure polysaccharides, levitic erythrocytes, viruses, bacteria, , Complement activation triggered by lipopolysaccharide (LPS) of parasites and damaged cells, which is traditionally known to result from spontaneous proteolytic production of C3b from complement factor C3.

As used herein, the term " lectin pathway " refers to complement activation that occurs through the specific binding of serum to non-serum carbohydrate-binding proteins, including mannan-binding lectin (MBL) and picoline.

As used herein, the term "classical pathway" refers to complement activation triggered by an antibody that binds to foreign particles and requires binding of the recognition molecule C1q.

As used herein, the term "MASP-2 inhibitor" means any agent which binds to or directly interacts with MASP-2 and effectively inhibits MASP-2-dependent complement activation, (Including, for example, MBL, H-picoline, and N-acetylglucosamine) within their lectin pathway, including their MASP-2 binding fragments, natural and synthetic peptides, small molecules, soluble MASP-2 receptors, M-picoline, or L-picoline), but does not encompass antibodies that bind to such other recognition molecules. MASP-2 inhibitors useful in the methods of the invention may reduce MASP-2-dependent complement activation by greater than 20%, such as greater than 50%, such as greater than 90%. In one embodiment of the invention, the MASP-2 inhibitor reduces MASP-2-dependent complement activation by 90% or more (i.e., MASP-2 complement activation is only 10% or less).

As used herein, the term "antibody" encompasses antibodies and antibody fragments thereof derived from all antibody-producing mammals (e.g., primates including mouse, rat, levitt and human) and includes MASP-2 poly Peptides or portions thereof. Exemplary antibodies include polyclonal, monoclonal and recombinant antibodies; Multispecific antibodies (e. G., Bispecific antibodies); Human adaptive antibodies; Murine antibodies; Chimeric, mouse-human, mouse-primate, primate-human monoclonal antibody; And anti-idiotypic antibodies, and may be all intact molecules or fragments thereof.

As used herein, the term "antibody fragment" generally refers to a portion derived or related to a full-length anti-MAST-2 antibody comprising antigen binding or variable regions thereof. Examples of antibody fragments include multispecific antibodies formed from Fab, Fab ', F (ab) 2, F (ab') 2 and Fv fragments, scFv fragments, dimers, linear antibodies, single-chain antibody molecules and antibody fragments do.

As used herein, a "single-chain Fv" or "scFv" antibody fragment comprises the VH and VL domains of an antibody, wherein such domains are provided in a single polypeptide chain. Generally, the Fv polypeptide further comprises a polypeptide linker between the VH and VL domains, which allows the scFv to form the antigen binding of the desired structure.

As used herein, a "chimeric antibody" is a recombinant protein containing a variable domain and a complementary-determining region derived from a non-human species (e. G., Rodent) antibody, while the remainder of the antibody molecule is derived from a human antibody will be.

As used herein, "human adaptive antibody" is a chimeric antibody that contains a minimal sequence identifying a specific complement-determining region derived from a non-human immunoglobulin transplanted into a human antibody framework. The human adaptive antibody is generally a recombinant protein, and only the complementary-determining region of the antibody is of non-human origin.

As used herein, the term "mannan-binding lectin" ("MBL") is synonymous with mann-binding protein ("MBP").

As used herein, a "membrane attack complex" ("MAC") is a complex of five terminal complement components (C5-C9) that are inserted and destroyed in the membrane. See C5b-9.

As used herein, a "subject" includes all mammals, including, without limitation, humans, non-human primates, dogs, cats, horses, sheep, goats, cows, rabbits, pigs and rodents. As used herein, amino acid residues are abbreviated as follows: alanine (Ala; A), asparagine (Asn; N), aspartic acid (Asp; D), arginine C), glutamic acid (Glu), glutamine (Gln), glycine (GI), histidine (H), isoleucine (Ile), leucine (L), lysine (Lys; K), methionine (Met; M), phenylalanine (Phe), proline (P), serine (S), threonine (Thr), tryptophan (Trp) ), And valine (Val; V).

At its best, the naturally occurring amino acids can be divided into groups based on the chemical properties of the side chains of each amino acid. "Hydrophobic" amino acids means lie, Leu, Met, Phe, Trp, Tyr, VaI, Ala, Cys or Pro. "Hydrophilic" amino acid means GIy, Asn, GIn, Ser, Thr, Asp, GIu, Lys, Arg or His. These amino acid classes can be further divided into sub-units as follows. "Amorphous hydrophilic" amino acids are Ser, Thr, Asn, or GIn. An "acid" amino acid is GIu or Asp. A "basic" amino acid is Lys, Arg or His.

As used herein, the term "conservative amino acid substitution" means substitution between amino acids within each of the following groups: (1) glycine, alanine, valine, leucine, and isoleucine, (2) phenylalanine, tyrosine, , (3) serine and threonine, (4) aspartate and glutamate, (5) glutamine and asparagine, and (6) lysine, arginine and histidine.

The term "oligonucleotide" as used herein refers to an oligomer or polymer of ribonucleic acid (RNA) or deoxyribonucleic acid (DNA) or a mimic thereof. The term also includes oligonucleotide bases composed of oligonucleotides that include naturally occurring nucleotides, covalent (skeletal) linkages between sugars and nucleosides, and non-naturally occurring modifications.

II. Alternative Paths: New Understanding

The complement alternative pathway was first described by Louis Pillemer and his colleagues in the early 1959 based on the study that gemic acid prepared from yeast cell walls is used to activate complement (Pillemer, L. et al., J. Exp. Med. 103: 1-13,1956, Lepow, IH, J. Immunol. 125: 471-478, 1980). Since then, gymosan has been considered as a normative example of a specific activator of alternative pathways in human and rodent sera (Lachmann, PJ, et al., Springer Semin. Immunopathol. 7: 143-162, 1984; Van Dijk, H., et al., J. Immunol. Methods 85: 233-243, 1985; Pangburn, MK, Methods in Enzymol. 162: 639-653, 1988). A common widely used measure of alternative pathway activation is to incubate serum with germic acid coated on plastic wells and to measure the amount of C3b adhered to the solid phase after incubation. As expected, there was substantial C3b attachment on the zymosan-coated well after incubation with normal mouse serum (Fig. 7B). However, when sera from homozygous MASP-2-deficient mice were cultured in zymosan-coated wells, C3b attachment was substantially reduced compared to normal serum. Additionally, the use of sera of mouse heterozygosity for deficiency in the MASP2 gene in this assay indicates the level of C3b attachment, the intermediate between the allogeneic MASP-2-deficient mouse serum and those obtained in normal mouse serum. Was obtained using another polysaccharide coated well known to activate the alternative pathway, which met the same results (Fig. 7A). This unexpected result demonstrates that MASP-2 plays an essential role in the activation of alternative pathways, since normal and MASP-2 deficient mice share the same genetic background, except for the MASP2 gene.

These results provide strong evidence that alternative pathways are independent of complement activation and are not self-acting pathways, as is essential for current research on current medical textbooks and complementation. In current, widely subscribing scientific papers, the alternative pathway is activated on the surface of a specific particle targeting target (microbe, zymosan, levitic erythrocytes) through amplification of spontaneous "tick-over" C3 activation. However, the absence of significant alternate pathway activation in serum of MASP-2 knockout mice by both known alternative pathway "activators " makes it invisible to account for the important physiological mechanisms of complement activation with" .

Since the MASP-2 protease is known to possess a specific and well-defined role as an enzyme responsible for the initiation of the lectin complement cascade, this result is a critical first step in the activation of the subsequent alternative pathway, Which is involved in the activation of the lectin pathway. C4b is the active product produced by the lectin pathway, not by the alternative pathway. Consistent with this concept, incubation of normal mouse serum with zymosan- or mannan-coated wells results in C4b attached to the wells, and when the coated wells are cultured in serum of MASP-2- deficient mice, this C4b attachment is substantially (Figs. 6A, 6B and 6C).

Alternative pathways may provide amplification loops for early activation of complement activation through classical and lectin pathways, with a well known role as an independent pathway of complement activation (Liszewski, MK and JP Atkinson, 1993, in Fundamental Immunology , Third Edition, edited by WE Paul, Raven Press, Ltd., New York; Schweinie, JE, et al., J. Clin. Invest. 84: 1821-1829, 1989). This alternative pathway-mediated amplification mechanism, the C3 convertase (C4b2b) generated by activation of each of the classical or lectin complement cascades, cleaves C3 to C3a and C3b and thus can participate in the formation of C3bBb, Gt; C3b < / RTI >

A possible explanation for the absence of alternative pathway activation in MASP-2 knockout serum is that the lectin pathway is required for early complement activation by "activators" of zymosan, mannan, and other potential alternative pathways, Plays a crucial role in amplifying complement activation. In other words, the alternative path is not a non-dependent linear cascade but rather a lectin for activation and a classical complement path-dependent feedforward amplification loop.

Rather than the complement cascade activated through two distinct paths (classical, alternate, and lectic paths) as described above, our results are more accurate to observe the complement composed of the two main systems, with a first approximation, It corresponds to the congenital (lectin) and acquired (classic) wings of the immune defense system. Lectins (MBP, M-picoline, H-picoline, and L-picoline) are specific recognition molecules that trigger the congenital complement system, which includes the lectin pathway and cannulated alternative pathway amplification loop. C1q is a specific recognition molecule that triggers the acquired complement system, which includes a classical pathway and an alternative pathway amplification loop. We call these two major complement activation systems a lectin-dependent complement system and a C1q-dependent complement system, respectively.

Along with the essential role in immune defense, the complement system contributes to the tissue damage of many clinical symptoms. Thus, there is an increasing need to develop therapeutically effective complement inhibitors to prevent such adverse effects. It has been recognized that it is highly desirable to specifically inhibit only the complement activation system which causes certain pathological conditions in which the possibility of immune defense of the complement is not completely blocked in the recognition that the complement consists of two main complement activation systems. For example, in a disease state with complement activation mediated predominantly by a lectin-dependent complement system, it is advantageous to specifically inhibit only this system. This will not compromise the C1q-dependent complement activation system, regulating immune complex processing and assisting host defense against infection.

A preferred protein component that is targeted for the development of therapeutics that specifically inhibit the lectin-dependent complement system is MASP-2. In all protein components of the lectin-dependent complement system (MBL, H-picoline, M-picoline, L-picoline, MASP-2, C2-C9, factor B, factor D, Are unique to a lectin-dependent complement system and are required to function as a system. Lectins (MBL, H-picoline, M-picoline and L-picoline) are the only components in the lectin-dependent complement system. However, the loss of any one of the lectin components does not necessarily inhibit activation of the system due to lectin ingestion. It is necessary to inhibit all four lectins to ensure inhibition of the lectin-dependent complement activation system. In addition, since MBL and picolin are known to possess complement independent opsonin activity, inhibition of lectin function results in loss of host defense mechanisms against this beneficial infection. In contrast, when MASP-2 is an inhibitory target, this complement-independent lectin opsonin activity remains intact. An additional benefit of MASP-2 as a therapeutic target to inhibit the lectin-dependent complement activation system is that the plasma concentration of MASP-2 is the lowest among all complement proteins (~ 500 ng / ml); Thus, a low concentration of high affinity inhibitor of MASP-2 would be needed to achieve complete inhibition (Moller-Kristensen, M., et al, J. Immunol Methods 282: 159-167, 2003).

III. The role of MASP-2 in various diseases, symptoms and treatment methods using MASP-2 inhibitors

Ischemic reperfusion injury

Ischemia reperfusion injury (I / R) occurs when blood flow is restored after ischemia. This is a common cause of morbidity and mortality within a broad spectrum of diseases. Surgery patients are treated for aortic aneurysm repair, cardiopulmonary bypass surgery, such as vascular reassortment related to organ transplantation (eg, heart, lung, liver, kidney) and finger / limb relapse, stroke, myocardial infarction, It is vulnerable after resuscitation and / or surgical procedures. Patients with atherosclerotic disease are prone to myocardial infarction, stroke, and embolism-induced gut and lower limb ischemia. Trauma patients often undergo transient ischemia of the extremities. In addition, the cause of all massive blood loss leads to systemic I / R responses.

The pathophysiology of I / R injury is a complex of at least two key contributors to progression: neutrophil stimulation with complement activation and oxygen radical-mediated damage. In I / R injury, complement activation was first described in myocardial infarction thirty years ago and has been extensively investigated in the complement system for I / R tissue injury (Hill, JH, et al., J. Exp. Med. 133: 885-900, 1971). Accumulated evidence points to complement as a major mediator of I / R damage. Complement suppression has been successful in the limited impairment of some I / R animal models. In an early study, C3 depletion was achieved with the injection of a cobranotoxin and was reported to be beneficial in I / R in the kidney and heart (Maroko, PR, et al., 1978, J. Clin Invest. 61: 661-670, Stein, SH, et al., Miner Electrolyte Metab. 11: 256-61, 1985). However, the soluble form of complement receptor 1 (sCR1) was the first complement-specific inhibitor used to prevent myocardial I / R damage (Weisman, H. F., et al., Science 249: 146-51, 1990). SCR1 treatment of myocardial I / R weakens the infarction associated with decreased leukocyte infiltration after reperfusion and a decrease in C5b-9 complex attachment of the cardiac endothelium.

In experimental myocardial I / R, the C1 esterase inhibitor (C1 INH) administered prior to reperfusion prevents the adhesion of C1q and significantly reduces cardiac muscle necrosis (Buerke, M., et al., 1995, Circulation 91: 393 -402, 1995). Animals with genetic deficiencies of C3 were less localized necrosis after skeletal muscle or intestinal ischemia (Weiser, M.R., et al., J. Exp. Med. 183: 2343-48, 1996).

Membrane attack complexes were the ultimate carrier of complement-directed damage, and studies of C5-deficient animals show reduced local and remote damage within the I / R injury model (Austen, WG Jr., et al., Surgery 126: 343-48,1999). The complement activation inhibitor, soluble Crry (complement receptor-related gene Y), has been shown to be effective for damage before and after the onset of murine reperfusion injury (Rehrig, S., et al., J. Immunol. 167: 5921-27, 2001). In the skeletal muscle ischemic model, the use of soluble complement receptor 1 (sCR1) reduced muscle damage when supplied after initiation of reperfusion (Kyriakides, C, et al., Am. J. Physiol., Cell Physiol. , 2001). In a pig model of myocardial I / R, animals treated with monoclonal antibody ("MoAb ") against anapylatoxin C5a prior to reperfusion showed reduced infarction (Amsterdam, EA, et al., Am. J. Physiol Heart Circ Physiol 268: H448-57, 1995). C5 MoAb treated rats reduced myocardial infarct size, neutrophil infiltration and apoptosis (Vakeva, A., et al., Circulation 97: 2259-67, 1998). These results highlight the importance of complement activation in the pathogenesis of I / R injury.

It is not clear that the complement pathway (classical, lectin or alternative) is predominantly involved in complement activation in I / R damage. Weiser et al. Demonstrate the important role of lectins and / or classical pathways in skeletal I / R by showing that C3- or C4-knockout mice are protected against I / R damage based on a significant reduction in vascular permeability (Weiser, MR, et al., J. Exp. Med. 183: 2343-48, 1996). In contrast, kidney I / R experiments with C4 knockout mice showed no significant tissue protection, whereas C3-, C5-, and C6-knockout mice were protected from damage, Suggesting that activation occurs through alternative pathways (Zhou, W., et al., J. Clin. Invest. 105: 1363-71, 2000). Using factor D deficient mice, the literature suggests that Stahl et al. Have recently played an important role in alternative pathways in the mouse intestinal I / R (Stahl, G., et al., Am. J. Pathol. 162 : 449-55, 2003). In contrast, Williams et al. Have shown a dominant role in the classical pathway for the initiation of mouse intestinal I / R damage by showing reduced C3 long-term staining and protection from damage of C4 and IgM (Ragl - / -) deficient mice (Williams, JP, et al., J. Appl. Physiol. 86: 938-42, 1999).

Treatment of myocardial I / R model with a monoclonal antibody to retmannan-binding lectin (MBL) reduces reperfusion injury after ischemia (Jordan, J.E., et al., Circulation 104: 1413-18, 2001). MBL antibody reduces complement adherence of endothelial cells in vitro after oxidative stress and represents the role of the lectin pathway in myocardial I / R damage (Collard, CD, et al., Am. J. Pathol. 156: 1549-56 , 2000). There is evidence that some organs of I / R injury can be mediated by a specific category of IgM, which is the activation of natural antibodies and classical pathways (Fleming, SD, et al., J. Immunol. 169: 2126-33 , 2002; Reid, RR, et al., J. Immunol. 169: 5433-40, 2002).

Several complement activation inhibitors have been developed as potential therapeutic agents to prevent morbidity and mortality due to myocardial I / R complications. Two of these inhibitors, sCR1 (TP10) and human adaptive anti-C5 scFv (pepticir major), completed Phase II clinical trial II. Pegselizumab completed an additional phase III clinical trial. TP10 was well tolerated and beneficial in the I / II study, but did not achieve the primary goal in February 2002 at the end of Phase II. However, a good analysis of data from high-risk male patients undergoing open-heart surgery has shown a significant reduction in mortality and infarct size. Administration of the human adaptive anti-C5 scFv reduced the overall patient acute myocardial infarction mortality in COMA and COMPLY II trials, but did not achieve the primary goal (Mahaffey, KW, et al., Circulation 108: 1176-83 , 2003). Results of a Phase III-C5 scFv clinical trial (PREVIO-CABG) to improve the surgically induced outcome after cardiac artery bypass have recently been published. Although the primary goal of this study was not achieved, this study demonstrated a general reduction in postoperative morbidity and mortality.

Dr. Walsh and co-workers have demonstrated that MBL is absent to avoid activation of the MBL-dependent lectin pathway, but mice with fully-activated classical complement pathways are consequently protected from cardiac reperfusion injury while preserving cardiac function (Walsh et al. , J. Immunol. 775: 541-46, 2005). Significantly, deficiency of C1q, a recognition component of the classical complement pathway, failed to protect mice with the intact MBL complement pathway from damage. These results indicate that the lectin pathway plays a major role in the pathogenesis of myocardial reperfusion ischemic injury.

Complement activation is known to play an important role in tissue damage associated with gastrointestinal ischemia - reperfusion (I / R). Using a GI / R murine model, Hart and colleagues recently reported that mice genetically deficient in MBL were protected from intestinal damage after gastrointestinal I / R (Hart et al., J. Immunol. 774: 6373 -80, 2005). Addition of recombinant MBL to MBL-deficient mice significantly increases the damage compared to untreated MBL-deficient mice after gastrointestinal I / R. In contrast, mice lacking C1q, a genetically classical pathway recognition component, were not protected from tissue damage following gastrointestinal I / R.

Renal I / R is a major cause of acute renal failure. The complement system appears to be involved in kidney I / R damage. In a recent study, de Vries and colleagues reported that the lectin pathway is activated at the experimental and clinical stages of renal I / R injury (de Vries et al., Am. J. Path. 165: 1677-88, 2004 ). Moreover, the lectin pathway proceeds and is co-localized with complement C3, C6, and C9 attachments in the kidney I / R. These results imply that they are involved in lectin pathway renal I / R damage of complement activation.

One aspect of the present invention relates to the treatment of ischemic reperfusion injury by treating a patient with ischemic reperfusion in a therapeutically effective amount of a MASP-2 inhibitor in a pharmaceutical carrier. MASP-2 inhibitors are administered to a subject by arterial, intravenous, intracranial, intramuscular, subcutaneous, or other parenteral administration, and orally, and most suitably by an arterial or intravenous administration of potentially non-peptide inhibitors . Administration of the MASP-2 inhibiting composition of the present invention is preferably performed immediately or as soon as possible after ischemia reperfusion. MASP-2 inhibitors may be administered before and / or after reperfusion and / or after reperfusion if the reperfusion occurs under controlled conditions (e.g., aortic aneurysm repair, organ transplantation or severe traumatic limb or finger rejoining). Administration may be repeated periodically as determined by the clinician for optimal therapeutic effect.

Atherosclerosis

There is considerable evidence that complement activation is associated with the development of atherosclerosis in humans. Numerous studies have shown that complement is widely active in atherosclerotic lesions and is particularly strong in vulnerable, ruptured plaques, although significant complement activation does not occur in the normal arteries. The components of the terminal complement pathway frequently occur in human atheroma (Niculescu, F., et al., Mol. Immunol. 36: 949-55.10-12, 1999; Rus, HG, et al., Immunol. Lett. 20: 305-310, 1989. Torzewski, M., et al., Arterioscler. Thromb. Vase. Biol., 18: 369-378, 1998). C3 and C4 adherence in arterial lesions has been demonstrated (Hansson, G. K., et al., Acta Pathol. Microbiol. Immunol. Scand. (A) 92: 429-35, 1984). The degree of C5b-9 adhesion was found to correlate with the severity of lesions (Vlaicu, R., et al., Atherosclerosis 57: 163-77, 1985). Attachment of non-C5b-9 complement iC3b is particularly robust in rupture and vulnerable plaques suggesting that complement activation may be a factor in acute cardiac syndrome (Taskinen S., et al., Biochem. J. 367: 403 -12, 2002). In experimental rabbit atheroma, complement activation is known to promote the development of lesions (Seifer, PS, et al., Lab Invest. 60: 747-54, 1989).

In atherosclerotic lesions, complement is activated through classical and alternative pathways, but evidence of complement activation through the lectin pathway is rarely present. Some components of the arterial wall can trigger complement activation. The classical pathway of complement may be activated by C-reactive protein (CRP) bound to enzymatically degraded LDL (Bhakdi, S., et al., Arterioscler. Thromb. Vase. Biol. 19: 2348-54 , 1999). Consistent with this study is the finding that terminal complement proteins are co-localized to the CRP in the lining of early human lesions (Torzewski, J., et al., Arterioscler. Thromb. Vase. Biol. 18: 1386-92, 1998) . Thus, immunoglobulin M or IgG antibodies specific for oxidized LDL in lesions can activate the classical pathway (Witztum, J. L., Lancet 344: 793-95, 1994). Lipids isolated from human atherosclerotic lesions have high levels of unesterified cholesterol and can activate alternative pathways (Seifert P., et al., J. Exp. Med. 172: 547-57, 1990). Pneumonia Chlamydia, a frequently associated Gram-negative bacteria with atherosclerotic lesions, can activate an alternative pathway of complement (Campbell L. A., et al., J. Infect. Dis. 172: 585-8, 1995). Other potential complement activators present in atherosclerotic lesions include cholesterol crystals and cellular debris, both of which may activate an alternative pathway (Seifert, PS, et al., Mol. Immunol. 24: 1303- 08, 1987).

By-products of complement activation are known to possess a number of biological properties that influence the development of atherosclerotic lesions. Local complement activation can induce cell lysis and produce at least some of the cellular debris found at the necrotic center of advanced lesions (Niculescu, F. et al., Mol. Immunol. 36: 949-55. ). Low soluble complement activation may be a significant contributor to smooth muscle cell proliferation and mononuclear infiltration into the intima of the artery during atherosclerosis (Torzewski J., et al., Arterioscler., 18: 673-77 , 1996). Consistent activation of complement may be crucial because it induces persistent inflammation. Along with infiltration of the complement component of blood plasma, arterial cells express the messenger RNA of the complement protein and the expression of various complement components is upregulated in atherosclerotic lesions (Yasojima, K., et al., Arterioscler. Thromb Vase Biol., 21: 1214-19, 2001).

A limited number of studies have been reported on complement protein deficiency in the development of atherosclerosis. The conclusion in the experimental animal model is controversial. In situ, the formation of atherosclerotic-like lesions induced by toxic doses of vitamin D was reduced in complement-deficient animals (Geertinger P., et al., Acta. Pathol. Microbiol. Scand. 284-88, 1970). In addition, in cholesterol-fed levet, the genetic deficiency of C6 (Geertinger, P., et al., Artery 1: 177-84, 1977; Schmiedt, W., et al., Arterioscl. Thromb. Complement inhibition by the K-76 COONa (Saito, E., et al., J. Drug Dev. 3: 147-54, 1990) It inhibits the development of arteriosclerosis. In contrast, recent studies have reported that C5 deficiency does not reduce the development of atherosclerotic lesions in apolipoprotein E (ApoE) deficient mice (Patel, S., et al., Biochem. Biophys. Res. Commun. 286: 164-70, 2001). However, another study has evaluated the development of atherosclerotic lesions in LDLR-deficient (Idir-) mice that are C3 deficient or not deficient (Buono, C, et al., Circulation 105: 3025-31, 2002). They found that mutation to atherosclerosis-like lesions of atheroma is partly dependent on the presence of an intact complement system.

One aspect of the present invention relates to treating or preventing atherosclerosis by treating a subject susceptible to or susceptible to atherosclerosis to a therapeutically effective amount of a MASP-2 inhibitor in a pharmaceutical carrier. The MASP-2 inhibitor may be administered to a subject by arterial, intravenous, intrathecal, intracranial, intramuscular, subcutaneous or other parenteral administration, and by oral administration of potentially non-peptic inhibitors. Administration of the MASP-2 inhibiting composition can be carried out prophylactically after the diagnosis of atherosclerosis in a subject or in a subject at high risk of developing such a condition. Administration may be repeated periodically as determined by the clinician for optimal therapeutic effect.

Other vascular diseases and symptoms

Endothelium is widely exposed to the immune system and is particularly susceptible to complement proteins present in plasma. Complement-mediated vascular injury may be caused by ischemia-reperfusion injury (Weisman, HF, Science 249: 146-51, 1990) and myocardial infarction (Seifert, PS, et al., Atherosclerosis 73: Tada, T., et al., Virchows Arch 430: 327-332, 1997). Evidence suggests that complement activation may extend to other vascular events.

For example, there is evidence that complement activation contributes to the pathogenesis of multiple vasculitides: vascular inflammation associated with rheumatoid arthritis (so-called malignant rheumatoid arthritis), systemic lupus erythematosus, systemic lupus erythematosus, , And Takayasu bottles. Henoch-Shenline spinal nephritis is a form of systemic vasculitis of the small blood vessels with an immunological pathogenesis, and complement activation which induces C5b-9-induced endothelial damage through the lectin pathway is recognized as an important mechanism (Kawana, S. et al. Endo, M., et al., Am. J. Kidney Dis. 35: 401-7, 2000). Systemic lupus erythematosus (SLE) is an example of systemic autoimmune disease affecting many organs such as the skin, kidney, joints, hepatic membrane surface, and central nervous system, and is frequently associated with severe vasculitis. IgG anti-endothelial antibodies and IgG complexes binding to endothelial cells are present in the serum of patients with active SLE, and adhesion of IgG immunoconjugates and complement are found within the vascular wall of patients with SLE vasculitis (Cines, DB, et al. , J. Clin. Invest., 73: 611-25, 1984). (Tomooka, K., Fukuoka Igaku Zasshi 80: 456-66, 1989), immune-complex vasculitis, vasculitis associated with hepatitis A, leukocytopenic vasculitis, and arteriosclerosis known as Takayama sickness Forms another polymorphic group of human diseases in which complement-dependent cytotoxicity to endothelium and other cell types plays a documented role (Tripathy, NK, et al., J. Rheumatol. 28: 805-8 , 2001).

There is evidence from the week that the complement activation plays a role in dilated cardiomyopathy. Expanded cardiomyopathy is a syndrome of heart swelling and impaired cardiac contractility. Recent data indicate that ongoing inflammation in the myocardium can contribute to the development of the disease. C5b-9, and complement adenylate complexes are known to have a significant correlation with immunoglobulin attachment and myocardial expression of TNF-alpha. In myocardial biopsies from 28 patients with dilated cardiomyopathy, C5b-9 myocardial accumulation was demonstrated, suggesting that chronic immunoglobulin-mediated complement activation in the myocardium partially contributes to the progression of dilated cardiomyopathy (Zwaka, TP, et al Am. J. Pathol, 161 (2): 449-57, 2002).

One aspect of the present invention is to provide a method of treating cardiovascular, cerebrovascular, peripheral (e.g., musculoskeletal) vascular, renal vascular, and mesenteric / intestinal vascular symptoms by administering to a pharmaceutical carrier a composition comprising a therapeutically effective amount of a MASP- Which comprises administering a therapeutically effective amount of a compound of the invention. Symptoms that may be considered suitable for the present invention include, but are not limited to: vasculitis such as Henoch-Shenline spinal nephritis, systemic lupus erythematosus-associated vasculitis, vasculitis associated with rheumatoid arthritis (so-called malignant rheumatoid arthritis), immune complex vasculitis and Takayama sickness; Dilated cardiomyopathy; Diabetic angiopathy; Kawasaki disease (arteritis); And venous air embolism (VGE). In addition, it has been given that complement activation occurs as a result of inflammatory response to exogenous substances associated with endotracheal trauma and cardiovascular invasion processes, and the MASP-2 inhibitory composition of the present invention can be used for restenosis after stent insertion, Percutaneous transluminal coronary angioplasty (PTCA), alone or in combination with other restenosis inhibitors, is known in the art, Patent No. 6,492,332 (Demopulos).

The MASP-2 inhibitor may be administered to a subject by arterial, intravenous, intramuscular, intrathecal, intracranial, subcutaneous or other parenteral administration, and by oral administration of potentially non-peptic inhibitors. Administration may be repeated periodically as determined by the clinician for optimal therapeutic effect. For inhibition of restenosis, the MASP-2 inhibitory composition may be administered prior to and / or during and / or after the stent implantation or atherectomy or angiogenesis. Alternatively, the MASP-2 inhibitory composition may be coated on or incorporated into the stent.

Gastrointestinal disorder

Ulcerative colitis and Crohn's disease are chronic inflammatory disorders of the bowel belonging to the area of inflammatory bowel disease (IBD). IBD is characterized by naturally occurring, chronic, recurrent inflammation of unknown origin. Despite the careful study of both human and experimental animal diseases, the precise mechanism of pathology is still unknown. However, the complement system is known to be activated in IBD patients and is known to play a role in the pathogenesis of disease (Kolios, G., et al., Hepato-Gastroenterology 45: 1601-9, 1998; Elmgreen, J., Dan. Med Bull 33: 222, 1986).

C3b and other activated complement products have been found in the intracapsular surface of epithelial cells of the surface of IBD patients and intramuscular and submucosal vessels (Halstensen, TS, et al., Immunol Res. 10: 485-92, 1991; Halstensen, TS, et al., Gastroenterology 98: 1264, 1990). In addition, the results of polymorphonuclear cell infiltration, generally C5a production, are observed as lymphocytes in inflammatory bowel movements (Kohl, J., MoI. Immunol. 38: 175, 2001). Multifunctional complement inhibitor K-76 has been described in a small clinical trial (Kitano, A., et al., Dis. Colon Rectum 35: 560, 1992), and carrageenan-induced colitis leptis model (Kitano, A., et al. , Clin. Exp. Immunol. 94: 348-53, 1993).

New human C5a receptor antagonists appear to be protected against the disease pathology of the retmodel of IBD (Woodruff, TM, et al, J. Immunol. 171: 5514-20, 2003). Disruption-accelerating factor (DAF), a genetically deficient mouse within the membrane complement regulatory protein, is used in IBD models to significantly increase tissue damage and increase proinflammatory cytokine production (Lin, F., et al. , J. Immunol., 172: 3836-41, 2004). Therefore, the complement control of the complement is important for regulating intestinal homeostasis and may be a major pathogenic mechanism involved in the development of IBD.

The present invention thus includes compositions comprising a therapeutically effective amount of a MASP-2 inhibitor in a pharmaceutical carrier, including pancreatitis, diverticulitis and bowel disorders such as Crohn's disease, ulcerative colitis, and irritable bowel syndrome by administering to a patient suffering from such a disorder Dependent < / RTI > complement activation in a subject suffering from an inflammatory gastrointestinal disorder that is not limited thereto. MASP-2 inhibitors may be administered to a subject by arterial, intravenous, intramuscular, subcutaneous, intrathecal, intracranial, or other parenteral administration, and orally, potentially non-peptide inhibitors. Administration may be repeated periodically as determined by the clinician for optimal therapeutic effect.

Pulmonary symptoms

Complications include acute respiratory distress syndrome (ARDS) (Ware, L, et al., N. Engl. J. Med. 342: 1334-49, 2000); Transfusion-associated acute lung injury (TRALI) (Seeger, W., et al., Blood 76: 1438-44, 1990); Ischemia / reperfusion acute lung injury (Xiao, F., et al., J. Appl. Physiol. 82: 1459-65, 1997); Chronic obstructive pulmonary disease (COPD) (Marc, M.M., et al., Am. J. Respir. Cell Mol. Biol. (Epub ahead of print), March 23, 2004); Asthma (Krug, N., et al., Am. J. Respir. Crit Care Med. 164: 1841-43, 2001); Wegener's granulomatosis (Kalluri, R., et al., J. Am. Soc. Nephrol. 8: 1795-800, 1997); And an anti-glomerular basement membrane disease (Goodpasture's disease) (Kondo, C, et al., Clin. Exp. Immunol. 124: 323-9, 2001).

It is now accepted that many ARDS pathophysiologies are involved in uncontrolled inflammatory cascades that are initiated as normal responses to infection or other stimuli, but ultimately cause significant host self-injury (Stanley, TP, Emerging Therapeutic Targets 2: 1 -16, 1998). ARDS patients show almost complete evidence of comprehensive complement activation (increased plasma levels of complement components C3a and C5a), and the degree of complement activation correlates with the development and outcome of ARDS (Hammerschmidt, DF, et al. , Lancet 1: 947-49, 1980; Solomkin, JS, et al., J. Surgery 97: 668-78, 1985).

Various experimental and clinical data indicate that it plays a role of complement activation in ARDS pathophysiology. In animal models, systemic activation of complement leads to acute lung injury with histopathology similar to that shown in human ARDS (Till, GO, et al, Am. J. Pathol. 129: 44-53, 1987; Ward, PA Am. J. Pathol., 149: 1081-86,1996). Inhibition of complement cascade by general complement depletion or specific inhibition of C5a leads to protection in acute lung injury animal models (Mulligan, M.S., et al., J. Clin. Invest. 98: 503-512, 1996). In rett models, sCR1 retains protective effects on complement- and neutrophil-mediated lung injury (Mulligan, M.S., Yeh, et al., J. Immunol. 148: 1479-85, 1992). In addition, virtually all complement components can be produced locally in the lungs by type II corneal cells, acinar macrophages and lung fibroblasts (Hetland, G., et al., Scand. J. Immunol. 24: 603-8 Rothman, B. L., et al., J. Immunol. 145: 592-98, 1990). The complement cascade thus contributes significantly to pulmonary inflammation and, as a consequence, to lung injury by ARDS.

Asthma is essentially an inflammatory disease. The basic features of allergic asthma include airway hyperresponsiveness to a variety of specific and nonspecific stimuli, excessive airway mucus production, pulmonary eosinophilia, and elevated serum IgE levels. Although asthma is a multidisciplinary origin, it is generally known to occur in genetically sensitive individuals as a result of improper immunological responses to general environmental antigens. The complement system has been reported to be highly active in the human asthmatic lungs (Humbles, AA, et al., Nature 406: 998-01, 2002; van de Graf, EA, et al., J. Immunol. Methods 147: 241-50,1992). In addition, recent animal models and human data provide evidence that complement activation is an important mechanism contributing to the pathogenesis of disease (Karp, CL, et al., Nat. Immunol. 1: 221-26, 2000; Bautsch, W et al., J. Immunol. 165: 5401-5, 2000; Drouin, SM, et al., J. Immunol. 169: 5926-33, 2002; Walters, DM, et al, Cell Mol. Biol. 27: 413-18, 2002). The role of asthma in the lectin pathway was supported by studies using a murine model of chronic fungal asthma. Mice with a genetic deficiency in mannan-binding lectin develop a modified airway hyperresponsiveness compared to normal animals in this asthma model (Hogaboam, CM, et al., J. Leukoc. Biol. 75: 805-14, 2004). The complement may be activated through several pathways in asthma: (a) activation through a classical pathway resulting from the formation of an allergenic-antibody complex; (b) alternate path activation on the surface of the allergen; (c) activation of the lectin pathway through the involvement of carbohydrate structures on allergic substrates; And (d) cleavage of C3 and C5 by proteases released from inflammatory cells. Identification of the complement activation pathways involved in developing allergic asthma focuses on the development of new therapeutic strategies for this increasingly important disease, although many do not know much about the complexes that play a complementary role in asthma.

Numerous studies using animal models have demonstrated a crucial role for C3 and its cleavage product, C3a, in the development of allergic phenotypes. Drouin and colleagues used C3-deficient mice in the ovalbumin (OVA) / Aspergillus fumigatus asthma model (Drouin et al., J. Immunol. 167: 4141-45, 2001) . They found that when tested with allergen, C3 deficient mice exhibited significantly reduced AHR and lung eosinophilia compared to matched wild type control mice. Additionally, these C3-deficient mice possess a significantly reduced number of IL-4 producing cells and reduced Ag-specific IgE and IgGl responses. Taube and colleagues obtained similar results in the asthma OVA model by blocking complement activation at C3 and C4 levels using a soluble recombinant form of the mouse complement receptor Crry (Taube et al., Am. J. Respir. Crit. Care Med., 168: 1333-41, 2003). Humbles and colleagues investigated the role of C3a in eosinophil function by deleting C3aR in mice (Humbles et al., Nature 40 (5: 998-1001, 2000). Using the asthma OVA model, the development of AHR Drouin and colleagues (2002) used C3aR-deficient mice in the OVA / A fumigatus asthmatic model and found that they have reduced AHR, eosinophil use, TH2 cytosine (Drouin et al., J. Immunol. 169: 5926-7), which has been shown to be effective in reducing allergic responses to C3-deficient animals that are susceptible to catechin production, lung mucus secretion, and reduced Ag-specific IgE and IgG1 responses Bautsch and colleagues used the OVA model of allergic asthma (Bautsch et al., J. Immunol. 165: 5401-05, 2000) to perform a survey using C3aR naturally deficient guinea pigs , They were treated with an airway Index axis was observed significant protection.

Numerous recent studies using animal models have demonstrated the critical role of C5 and their cleavage product C5a in the development of allergic phenotypes. Abe and co-workers have reported evidence linking C5aR activation to airway inflammation, cytokine production and airway responsiveness (Abe et al., J. Immunol. 167: 4651-60, 2001). In these studies, inhibition of complement activation by soluble CR1, futhan (complement activation inhibitor) or synthetic hexa peptide C5a antagonist blocks inflammatory and airway responsiveness to methacholine. In studies using blocking anti-C5 monoclonal antibodies, Peng and co-workers have found that C5 activation is substantially responsible for airway inflammation and AHR in the asthma OVA model (Peng et al., J. Clin. Invest. 115: 1590-1600, 2005). In addition, Baelder and colleagues reported that blockade of C5aR in A. fumigatus asthma models substantially reduced AHR (Baelder et al., J. Immunol. 774: 783-89, 2005). In addition, blocking of both C3aR and C5aR significantly reduces airway inflammation, which is evidenced by a reduced number of neutrophils and eosinophils in the BAL.

Although the previous studies highlighted the importance of the complement factors C3 and C5 and their cleavage products in the pathogenesis of experimental allergic asthma, C3 and C5 are common to all three activation pathways, But did not provide information on the contribution of However, a recent study by Hogaboam and colleagues pointed out that the lectin pathway may play a major role in the pathogenesis of asthma (Hogaboam et al., J. Leukocytye Biol. 75: 805-814, 2004). These studies used carbohydrate-binding proteins that function as recognition moieties for activation of the mouse and rhe- tein complement pathway that are genetically deficient in mannan-binding lectin-A (MBL-A). In the model of chronic fungal asthma, MBL-A (+ / +) and MBL-A (- / -), A. fumigatus-sensitized mice were classified as A. fumigatus conidia. And examined at 4 and 28 days after the test. The AHR in the sensitized MBL-A (- / -) mice was significantly reduced at both time points after the conidia test compared with the sensitized MBL-A (+ / +) group. They showed that lung TH2 cytokine levels (IL-4, IL-5 and IL-13) were significantly reduced in A. fumigatus-sensitized MBL-A (- / -) mice compared to wild type mice at 4 days postconidia . These results indicate that MBL-A and the lectin pathway play a major role in the development and maintenance of AHR in chronic fungal asthma.

Recent clinical findings suggest that the relationship between development of asthma and the specific MBL gene polymorphism provides additional evidence that the lectin pathway plays an important pathological role in this disease (Kaur et al., Clin. Experimental Immunol 143: 414-19, 2006). MBL plasma concentrations vary from individual to individual, and this contributes primarily to genetic polymorphisms within the MBL gene. They found that individuals with at least one copy of the specific MBL gene polymorphism with up to two to four-fold increased MBL expression almost 5-fold increased the risk of developing bronchial asthma. Increases the severity of disease markers in bronchial asthma patients with MBL gene polymorphisms.

The features of the present invention are therefore applicable to the treatment of acute respiratory distress syndrome, transfusion-related acute lung injury, ischemia / reperfusion acute lung injury, chronic obstructive pulmonary disease, asthma, Wegener's granulomatosis, anti- glomerular basement membrane disease (gastroparesis), meconium aspiration syndrome, Inhibitors of MASP-2 inhibitors in a pharmaceutical carrier are administered to a subject suffering from pulmonary circulation disorders, including, without limitation, closed bronchitis syndrome, idiopathic pulmonary fibrosis, secondary acute lung injury of burn, non-cardiac pulmonary edema, transfusion-related respiratory depression, There is provided a method of treating a pulmonary circulation disorder by administering a composition comprising a therapeutically effective amount. MASP-2 inhibitors can be administered to a subject by systemic administration, such as by the administration of an arterial, intravenous, intrathecal, intracranial, intramuscular, subcutaneous or other parenteral administration, and potentially non-peptide inhibitors. The MASP-2 inhibitor composition may be combined with one or more additional therapeutic agents such as anti-inflammatory, antihistamines, corticosteroids or antibiotics. Administration can be repeated as determined by the clinician until symptoms improve.

Cardiopulmonary bypass

There are a number of medical processes during which the blood is being converted from the patient circulatory system (extracorporeal circulation system or ECC). This process includes hemodialysis, plasma exchange, leukocyte subspecies, ECMO, heparin-induced outermost oxygenated LDL deposition (HELP), and cardiopulmonary bypass (CPB). These processes expose blood or blood products to foreign surfaces that alter normal cellular function and homeostasis. In the leading studies (Craddock et al.), Complement activation has been identified as a possible cause of granulocytopenia during hemodialysis (Craddock, PR, et al., N. Engl. J. Med. 296: 769-74, 1977 ). The results of many studies from 1977 to the present indicate that many adverse effects experienced by hemodialysis or CPB patients are caused by activation of the complement system (Chenoweth, DE, Ann. NY Acad. ScL 516: 306- Transplanta 9: 36-37, 1987; Hugli, TE, Complement 3: 111-127, 1986; Cheung, AK, J. Am. Soc. Nephrol 1: 150-161, 1990. Johnson, R. Nephrol Dial. 45, 1994). For example, complement activity is an important criterion for determining the biocompatibility of a hemodialyzer in terms of renal function recovery, susceptibility to infection, pulmonary dysfunction, morbidity, and survival in patients with impaired renal function (Hakim, RM, Kidney Int. : 484-4946, 1993).

It is widely known that complement activation by hematopoietic membrane is caused by an alternative pathway mechanism due to the production of weak C4a (Kirklin, JK, et al., J. Thorac., 86: 845-57, 1983, Vallhonrat, H., et al., ASAIO J. 45: 113-4, 1999), but recent studies have suggested that classical pathways may be involved (Wachtfogal, YT, et al., Blood 73: 468-471, 1989). However, there are still inadequate solutions to factors that initiate and regulate complement activation on artificial surfaces, including biomedical polymers. For example, Cuprophan membranes used for hemodialysis have been classified as very powerful co-activators. While not wishing to be bound by theory, the inventors have theorized that they may be partly explained by their polysaccharide properties. The MASP-2-dependent complement activation system identified in the present invention provides a mechanism by which activation of the lectin pathway triggers alternative pathway activation.

Patients with ECC in CPB have a systemic inflammatory response, which is partly caused by exposure to the artificial surface of the cardiopulmonary circuit of the blood, but also by surface-independent factors such as surgical trauma and ischemia-reperfusion injury Asimakopoulos, G., Perfusion < / RTI > (Suppl): S12-6, 1998; Asimakopoulos, G., < RTI ID = 0.0 & 14: 269-77,1999). The CPB-triggered inflammatory reaction can lead to postoperative complications, usually "post-perfusion syndrome". This postoperative course is due to lack of cognition (Fitch, J., et al., Circulation 100 (25): 2499-2506, 1999), respiratory failure, bleeding disorders, renal dysfunction and, , S., et al., Chest 112: 676-692, 1997). CPB's cardiac bypass surgery leads to severe complement activation (E. Fosse, 1987) when compared to surgery with a significant degree of surgical trauma but no CPB. Thus, the first suspected cause of such CPB-related problems is inadequate complement activation during the bypass procedure (Chenoweth, K., et al., N. Engl. J. Med. 304: 497-503, 1981. P. Haslam, et al., Anaesthesia 25: 22-26, 1980; JK Kirklin, et al., J. Thorac. Cardiovasc. Surg. 86: 845-857, 1983; -103, 1988. J. Steinberg, et al., J. Thorac. Cardiovasc. Surg 106: 1901-1918, 1993). In the CPB circuit, the alternative complement pathway plays a role in significant complement activation, which is the result of the interaction between the blood and the artificial surface of the CPB circuit (Kirklin, JK, et al., J. Thorac. Cardiovasc. Surg., 86: Et al., ASAIO J. 45: 113-4, 1999). However, there is also evidence that the classical complement pathway is activated at CPB (Wachtfogel, Y. T., et al., Blood 73: 468-471, 1989).

Primary inflammatory substances are generated after activation of the complement system and include anapylatoxins C3a and C5a, opsonin C3b, and membrane attack complex C5b-9. C3a and C5a are potent stimulators of neutrophils, monocytes, and platelets (Haeffner-Cavaillon, N., et al., J. Immunol., 139: 794-9, 1987; Fletcher, MP, Rinder, CS, et al., Circulation 100: 553-8,1999), < RTI ID = 0.0 > . Activation of these cells leads to the release of proinflammatory cytokines (IL-1, IL-6, IL-8, TNF alpha), oxidized free radicals and proteases (Schindler, R., et al., Blood 76: 1631- Kawamura, T., et al., Can J. Anaesth., 40: 1016-21, 1993, Steinberg, JB et al., J. Thorac. Cardiovasc. Surg. 106: 1008-1, 1993, Finn, A., et al., J. Thorac. Cardiovasc Surg. 105: 234-41, 1993, Ashraf, SS , et al, J. Cardiothorac, Vase, Anesth, 11: 718-22, 1997). C5a is known to upregulate the adhesion molecules CDl Ib and CD18 of Mac-1 in polymorphonuclear cells (PMNs), and that degranulation of PMN induces pre-release inflammatory enzymes (Rinder, C, et al., Cardiovasc Pharmacol 114: Kinkade, JM, Jr., et al., Biochem. Biophys. Res. Commun. 114: 296-303, 1983; Lamb, NJ, et al, Crit. Care Med. 27: 1738-44, 1999; Fujie, K., et al., Eur. C5b-9 induces the expression of the adherent molecule P-selegentin (CD62P) on platelets (Rinder, CS, et al., J. Thorac. Cardiovasc. Surg. 118: 460-6, 1999) C5b-9 all induce surface expression of P-cerectin on endothelial cells (Foreman, KE et al., J. Clin. Invest. 94: 1147-55, 1994). These adhesion molecules are involved in the interaction between leukocytes, platelets and endothelial cells. Expression of adhesion molecules on activated endothelial cells is responsible for the separation of activated leukocytes, which mediate tissue inflammation and damage (Evangelista, V., Blood 1999; Foreman, KE, J. Clin. Invest. 1994; Lentsch , ≪ / RTI > AB, et al., J. Pathol 190: 343-8,2000).

This is the action of these complement activation products on neutrophils, monocytes, platelets and other circulating cells that can cause a variety of problems that can occur after CPB. Several complement inhibitors have been studied for potential applications in CPB. These include recombinant soluble complement receptor 1 (sCR1) (Chai, PJ, et al., Circulation 101: 541-6, 2000), human adaptive single chain anti-C5 antibody (h5G1.1-scFv or peptelizumab) (Rinder, CS., Et al., Circulation 100: 553, 1999), recombinant fusion hybrids of human membrane cofactor proteins and human degradation accelerators (CAB-2, Fitch, JCK, et al., Circulation 100: 3499-506 (Nilsson, B., et al., Blood 92: 1661-7, 1998) and the anti-Factor D MoAb (Fung, M., et al., J. Thoracic Cardiovasc. Surg. 122: 113-22, 2001). sCR1 and CAB-2 inhibit the classical and alternative complement pathways in the C3 and C5 activation steps. Compstatin inhibits all of the complement pathways in the C3 activation step, where h5G1.1-scFv is in the C5 activation step. The anti-factor D MoAb inhibits the alternative pathway in the C3 and C5 activation steps. However, no such complement inhibitor specifically inhibits the MASP-2-dependent complement activation system identified in the present invention.

Three prospective clinical trials to investigate the efficacy and safety of human adaptive monoclonal anti-C5 antibody (h5G1.1-scFv, pectelizumab), which reduces MI and mortality before and after surgery in CABG surgery. The results of the study have been reported (Verrier, ED, et al., JAMA 291: 2319-27, 2004). Compared with placebo, peckcelizumab did not significantly reduce the risk of death or multiple targets of MI within 2746 patients undergoing CABG surgery. However, there were statistically significant reductions in 30 days postoperatively in all 3099 patients who underwent CABG with or without valve surgery. This inhibits the production of C5a and sC5b-9, but does not affect the production of the other two potent complement inflammatory substances, C3a and opsonin C3b, because it inhibits the C5 activation step, which also leads to CPB-induced inflammation It is known to contribute to the reaction.

One aspect of the present invention is therefore to provide a method of treating hyperlipidemia, including hemodialysis, plasma exchange, leukapheresis, extracorporeal oxygen uptake (ECMO), heparin-induced outermost oxygenated LDL precipitation (HELP) Inflammatory response by treating a subject suffering from a cardiopulmonary process comprising a patient with a composition comprising a therapeutically effective amount of a MASP-2 inhibitor in a pharmaceutical carrier. MASP-2 inhibitor treatment according to the methods of the present invention is sometimes known to be useful for reducing or preventing cognitive impairment resulting from the CPB process. MASP-2 inhibitors may be administered to pre-operative and / or intra-operative and / or post-operative subjects by, for example, arterial, intravenous, intramuscular, subcutaneous or other parenteral administration. Alternatively, the MASP-2 inhibitor may be administered to a patient, for example, by infusing a MASP-2 inhibitor either through or through a tube or membrane through which blood circulates, or by injecting blood into a surface coated with a MASP- And can be introduced into the bloodstream of the subject during extracorporeal circulation.

Inflammatory and non-inflammatory arthritis and other musculoskeletal disorders

Activation of the complement system is associated with a variety of pathogenic mechanisms of rheumatic diseases; Rheumatoid arthritis (Mollnes, TE, et al., Arthritis Rheum. 29: 1359-64, 1986), osteoarthritis (Mollnes et al., Molec. Immunol. 36: 905-14, 1999), rheumatoid arthritis (SLE) (Molina, H., Current Opinion in Rheumatol. 14: 492-497, 2002). 1987) and Sjögren's syndrome (Sanders, ME, et al., J. Immunol. 138: 2095-9, 1987) and Behçet's syndrome (Rumfeld, WR, et al., Br. J. Rheumatol. ).

There is clear evidence that immune-complex-triggered complement activation is a major pathological mechanism that contributes to tissue damage in rheumatoid arthritis (RA). There are numerous reports that complement activation products were increased in the plasma of RA patients (Morgan, BP, et al, Clin. Exp. Immunol, 73: 473-478, 1988; Auda, G., et al., Rheumatol. Int 10: 185-189, 1990; Rumfeld, WR, et al, Br J. Rheumatol 25: 266-270, 1986). The complement activation products such as C3a, C5a, and sC5b-9 are found in inflamed rheumatoid joints and a direct correlation between the degree of complement activation and the severity of RA has been established (Makinde, VA, et al., Ann. Rheum 48: 302-306, 1989; Brodeur, JP, et al., Arthritis Rheumatism 34: 1531-1537, 1991). In both adult and juvenile rheumatoid arthritis, increased serum and lubrication levels of the alternative pathway complement activation product Bb relative to C4d (a marker of classical pathway activation) indicate that complement activation is predominantly mediated by alternative pathways (El-Ghobarey, AF et al., J. Rheumatology 7: 453-460, 1980; Agarwal, A., et al, Rheumatology 39: 189-192, 2000). The complement activation product can directly damage damaged tissues directly (via C5b-9) or indirectly mediate inflammation through the introduction of inflammatory cells by anapylatoxins C3a and C5a.

Animal models of experimental arthritis have been widely used to investigate the role of complement in the pathogenesis of RA. Complement depletion by the Cobra venom factor in the RA animal model prevented the development of arthritis (Morgan, K., et al., Arthritis Rheumat. 24: 1356-1362, 1981; Van Lent, PL, et al., Am. J Pathol, 140: 1451-1461, 1992). Intraarticular injection of soluble form of complement receptor 1 (sCR1), a complement inhibitor, inhibited inflammation in the RA reticulation model (Goodfellow, R. M., et al., Clin. Exp. Immunol. 110: 45-52, 1997). In addition, sCR1 inhibits the development and progression of retrocorne-induced arthritis (Goodfellow, R. M., et al., Clin Exp. Immunol. 119: 210-216, 2000). Availability CR1 inhibits the classical and alternative complement pathway in the C3 and C5 activation steps in the alternative pathway and the classical pathway, thus inhibiting the production of C3a, C5a and sC5b-9.

In the late 1970s, rodents were inoculated with heterologous type II collagen (CII, a major collagen component of human articular cartilage), leading to the development of autoimmune arthritis (collagen-induced arthritis, or CIA) (Courtenay, JS, et al., Nature 283: 666-68 (1980), Banda et al., J. of Immunol. 171: 2109-2115 (2003)). Autoimmune responses in susceptible animals involve complex combinations of factors including specific major histocompatibility complex (MHC) molecules, cytokines and CII-specific B- and T-cell responses (reviewed by Myers, LK, et al., Life Sciences 61: 1861-78,1997). Almost 40% of pure mating mice were observed to have complete complement deficiency of complement component C5 (Cinader, B., et al., J. Exp. Med. 72 (9: 897-902, 1964) The results of these studies indicate that C5 sufficiency is absolutely necessary for the development of CIA (Watson et al., 1987; Wang, Y., et al., J. Immunol., 164: 4340-4347, 2000. Further evidence of the importance of C5 and complement in RA is provided by the use of anti-C5 monoclonal antibodies (MoAbs). Prophylactic intraperitoneal administration of anti-C5 MoAbs in the CIA murine model almost completely prevented disease outbreaks, and treatment of active arthritis resulted in significant clinical benefit and mitigation of histologic diseases (Wang, Y., et al , Proc Natl Acad Sci USA 92: 8955-59, 1995).

Additional studies on the potential role of complement activation in disease pathogenesis mechanisms have been provided by studies using K / BxN T-cell receptor gene insertion mice, a recently developed inflammatory arthritis model (Korganow, AS, et al., Immunity 7 (9: 451-461, 1999). All K / BxN animals spontaneously develop autoimmune diseases with almost all clinical, histological and immunological characteristics (although not all) of human RA. Arthritis K / BxN mice were transfected with serum from a healthy animal, and arthritis was induced through arthritis-induced immunoglobulin transfer. Arthritic K / BxN mice (Ji, H., et al., Immunity 16: 157-68, 2002). Interestingly, the results suggest that the alternative pathway Indicating that activation is crucial while classical pathway activation is unnecessary, while the production of C5a is critical because both C5-deficient mice and C5aR-deficient mice are protected from disease development. Previous studies have shown that C5a receptor expression protects mice from arthritis by genetic abstraction (Grant, EP, et al., J. Exp. Med. 796: 1461-1471, 2002).

Human adaptive anti-C5 MoAb (5G1.1), which prevents human complement component C5 from splitting into its pre-inflammatory components, is administered by Alexion Pharmaceuticals, Inc. (New Haven, Connecticut) as a potential therapeutic agent for RA It is under development.

Both studies independently suggested that the lectin pathway promotes inflammation in RA patients through the interaction of MBL with specific IgG glycoforms (Malhotra et al., Nat. Med. 7: 237-243, 1995 ; Cuchacovich et al., J. Rheumatol. 23: 44-51, 1996). RA has been associated with a record increase in the IgG glycoform deficient in galactose (IgG0 glycoform) in the molecule of the Fc region (Rudd et al, Trends Biotechnology 22: 524-30, 2004). The percentage of IgGO glycoform is increased with disease progression and return to normal when the patient is alleviated. In vivo, IgGO is attached to the lubrication tissue, and MBL provides an increased level of lubricant in RA patients. Aggregated Agalactosyl IgG (IgGO) on RA-related clustered IgG can bind mannose-binding lectin (MBL) and activate the complement lectin pathway. In addition, a recent clinical study to observe allelic variants of MBL in RA patients indicates that MBL plays an inflammatory-enhancing role in the disease (Garred et al., J. Rheumatol. 27: 26-34, 2000). Thus, the lectin pathway may play an important role in the pathogenesis of RA.

Systemic lupus erythematosus (SLE) is an autoimmune disease of undetermined etiology that causes autoantibodies, the generation of immune complex circulation, and the activation of intermittent, uncontrollable complement systems. Although the origin of autoimmunity in SLE is poorly defined, there is considerable information that complement activation is involved as an important mechanism contributing to vascular injury in this disease (Abramson, SB, et al., Hospital Practice 33: 107-122, 1998). Activation of both the classical and alternative pathways of complement involvement in this disease and both C4d and Bb are sensitive markers of moderate to severe lupus disease activity (Manzi, S., et al., Arthrit. Rheumat. 39: 1178-1188 , 1996). Alternative complement pathway activation involves disease outbreaks in systemic lupus erythematosus during pregnancy (Buyon, J. P., et al., Arthritis Rheum. 35: 55-61, 1992). In addition, the lectin pathway may contribute to disease development, since autologous MBL antibodies have recently been identified in the serum of SLE patients (Seelen, MA, et al., Clin Exp. Immunol. 734: 335-343, 2003 ).

Immune complex-mediated activation of the complement through the classical pathway is known to be a mechanism by which tissue damage occurs within SLE patients. However, a genetic deficiency in the complement component of the classical pathway increases the risk of lupus and lupus-like disease (Pickering, M. C, et al., Adv. Immunol. 16: 227-324, 2000). SLE, or related syndrome occurs in patients with a complete deficiency of more than 80% of C1q, C1r / C1s, C4, or C3. This represents an apparent paradox in which the protective effects and adverse effects of complement in lupus harmonize.

The important activity of the classical pathway seems to promote circulation by the mononuclear predation system and the removal of immune complexes from the tissue (Kohler, P. F., et al, Am. J. Med. 56: 406-11, 1974). In addition, complement has recently been shown to play an important role in the removal and elimination of apoptosis (Mevorarch, D., et al., J. Exp. Med. 188: 2313-2320, 1998). The lack of classical pathway function allows the developmental cycle in which immune complexes or apoptotic cells accumulate in the tissues, cause inflammation and autoantigen release, stimulate autologous antibodies and more immune complex production, thereby triggering autoimmune reactions (Botto, M., et al., Nat. Genet. 19: 56-59, 1998; Botto, M., Arthritis Res. 3: 201-10, 2001). However, this "complete" deficiency state of the classical pathway component appears in one out of 100 SLE patients. Thus, in the majority of SLE patients, complement deficiency in classical pathway components does not contribute to disease pathogenesis, and complement activation may be an important mechanism contributing to the mechanism of SLE development. The fact that rare individuals with permanent genetic deficiencies within the classical pathway component develop SLE frequently at some point in their lifetime demonstrate the mechanism that can cause disease.

The results of SLE animal models support an important role for complement activation in disease pathogenesis mechanisms. Inhibition of C5 activation using blocking anti-C5 MoAb reduces proteinuria and renal disease in NZB / NZW F1 mice, SLE mouse models (Wang Y., et al., Proc. Natl. Acad. 8, 1996). In addition, anti-C5 MoAb mouse treatment, which was severely associated with immunodeficient disease transplanted with a cytopathic anti-DNA antibody, showed improvement in proteinuria and renal tissue histology with respect to survival benefit compared to the untreated control (Ravirajan, CT, et al., Rheumatology 43: 442-1, 2004). The alternative pathway plays an important role in the autoimmune disease manifestation of SLE, and the deficiency of Factor B in the inverse crossing of the MRL / Ipr model of the factor B-deficient mouse and SLE results in a change in the level of vasculitis, glomerular disease, C3 consumption and other autoantibodies (Watanabe, H., et al., J. Immunol. 164: 786-794, 2000). Human adaptive anti-C5 MoAb is under investigation as a potential therapeutic agent for SLE. This antibody prevents C5 from splitting into C5a and C5b. In Phase I trials no significant adverse effects were seen, and in-depth human trials are underway to test the efficacy of SLE (Strand, V., Lupus 70: 216-221, 2001).

The results of human and animal studies support the possibility that the complement system directly contributes to the pathogenesis of muscle regression atrophy. Human dystrophin biopsy studies have shown that C3 and C9 were attached to both necrotic and non-necrotic fibers within the dystrophin muscle (Cornelio and Dones, Ann. Neurol. 76: 694-701, 1984; Spuler and Engel, AG, Neurology 50 : 41-46,1998). Using a DNA microarray, Porter and co-workers have found gene expression of a number of complement-associated mRNAs that have been increased historically in dystrophin-deficient (mdx) mice consistent with the development of dystrophin disease (Porter et al., Hum Genet, 77: 263-72, 2002). Mutations in the human gene encoding dysporine and dermal muscle proteins have been identified as major risk factors for both types of skeletal muscle diseases, namely zone musculoskeletal atrophy (LGMD) and Miyoshi myopathy (Liu et al., Nat. Genet. : 31-6, 1998). Several models of mutant mice in dysporin have been developed and they have also developed progressive muscle regressive atrophy. Complement cascade activation was identified on the surface of non-necrotic muscle fibers in some LGMD patients (Spuler and Engel., Neurology 50: 41-46, 1998). In a recent study, Wenzel and colleagues found that both murine and human dysprosin-deficient muscle fibers lack a specific inhibitor of the complement inhibitor, CD33 / DAF, C5b-9 MAC (membrane attack complex) et al., J. Immunol. 775: 6219-25, 2005). In conclusion, dysporine-deficient necrotic myocytes are more sensitive to complement-mediated cell lysis. Wenzel and colleagues suggest that complement-mediated dissolution of skeletal muscle cells may be a major pathological mechanism involved in the development of LGMD and Miyoshi myopathy patients. Connolly and colleagues have studied the role of complement C3 in the pathogenesis mechanism of dy- / - mice, a kind of neurodegenerative atrophy model, laminin α2-deficient (Connolly et al., J. Neuroimmunol. 127: 2002). They found animals that genetically depleted both C3 and laminin α2, and found that the absence of C3 in the dy - / - model of muscle regression atrophy prolonged survival. In addition, double knockout (C37 - / -, dy - / -) mice showed stronger muscle strength than dy - / - mice. This study suggests that the complement system can directly contribute to the onset mechanism of this form of congenital degeneration atrophy.

One aspect of the present invention is to provide a method of treating osteoarthritis, rheumatoid arthritis, inflammatory rheumatoid arthritis, gout, neuropathic arthropathy, psoriatic arthritis, rigidity, and osteoarthritis by administering to a subject suffering from such a disorder a composition comprising a therapeutically effective amount of a MASP- Inflammatory arthritis and other musculoskeletal disorders including but not limited to spondylitis or other vertebral arthritis and crystalline arthropathy, muscle regression atrophy or systemic lupus erythematosus (SLE). MASP-2 inhibitors may be administered to a subject by systemic administration, for example, by the administration of an arterial, intravenous, intramuscular, subcutaneous or other parenteral administration, or potentially non-peptide. Alternatively, administration can be administered by local delivery, such as intra-articular injection. The MASP-2 inhibitor may be administered periodically at extended time intervals to modulate or treat chronic conditions and may be administered before, during and / or after acute trauma or injury, including surgical procedures performed on the joints, Or repeated administration.

Kidney symptoms

Activation of the complement system is involved in the pathogenesis of various renal diseases; Glomerular proliferative glomerulonephritis (IgA-Kidney disease, Burger's disease) (Endo, M., et al., Clin. Nephrology 55: 185-191, 2001), glomerulonephritis (Kerjashki, D., Arch B Cell Pathol. 35: 976-84, 1989), membranous proliferation (Kidney Int., 41: 933-1, 1992; Salant, DJ, et al., Kidney Int. 75: 294-300, 1979; Meri, S., et al., J. Exp. Med. 775: 939-50 , 1992), glomerulonephritis (acute glomerulonephritis after acute infection), cold globulin glomerulonephritis (Ohsawa, L, et al., Clin Immunol. 101: 59-66, Autoimmunity 11: 61-6, 1991), and Henoch-Schlenne spinal nephritis (Endo, M., et al., Am. J. Kidney Dis. 35: 401-407, 2000). The complement relevance of kidney disease has been studied for decades, but the exact role of the onset, development and dissolution of kidney disease is still under debate. Under normal conditions, complement contributes to the host, but improper activation and attachment of complement may contribute to tissue damage.

There is substantial evidence that glomerulonephritis, glomerulonephritis inflammation, is often initiated by immune complex attachment on glomerular or tubular structures that trigger complement activation, inflammation, and tissue damage. Kahn and Sinniah have demonstrated increased C5b-9 adhesion in the tubular basement membrane of biopsies taken from patients with various forms of glomerulonephritis (Kahn, T. N., et al., Histopath. 26: 351-6, 1995). C5b-9 adherence in the tubular epithelium / basement membrane structure correlates with plasma creatine levels in patients with IgA nephropathy (Alexopoulos, A., et al., Nephrol, Dial. Transplant 70: 1166-1172, 1995). Another study of membranous kidney disease demonstrated the relationship between clinical outcome and urinary sC5b-9 levels (Kon, S.P., et al., Kidney Int. 48: 1953-58, 1995). Increased levels of sC5b-9 are positively correlated with poor prognosis. Lehto et al. Measured the increased levels of the plasma membrane in the urine of patients with glomerulonephritis and CD59, a complement regulator that inhibits the membrane-attack complex of C5b-9 (Lehto, T., et al., Kidney Int. 47: 1403-11, 1995). Histopathologic analysis of these biopsy specimens of the same patient demonstrated the attachment of C3 and C9 proteins in the glomeruli, but the expression of CD59 in these tissues was reduced compared to that of normal kidney tissue. These various studies suggest that complement proteins, which are correlated with the degree of tissue damage and disease prognosis, are excreted in the urine by ongoing complement-mediated glomerulonephritis.

Glomerulonephritis Inhibition of complement activation in various animal models has demonstrated the importance of complement activation in the pathogenesis of the disease. In a model of proliferative glomerulonephritis (MPGN), anti-Thyl antiserum infusion in a C6-deficient rat (not forming C5b-9) reduced glomerular cellular proliferation by 90% compared to C6 + steady state, and platelet and macrophage infiltration (Brandt, J., et al., Kidney Int. 49: 335-343, 1996), which shows a reduced collagen type IV synthesis (marker of angiomatous enlargement) and a 50% . These results indicate that C5b-9 is involved as a major mediator of complement-mediated tissue damage in the ectopic anti-thymocyte serum model. As another model of glomerulonephritis, infusion of multistage doses of levitic antral-glomerular basement membrane produces a dose-dependent inflow of polymorphonuclear leukocyte (PMN), which is monitored by pretreatment of the cobra venom factor (Scandrett, AL, et al., Am. J. Physiol. 2: 58: F256-F265, 1995). Cobra venom-treated rats exhibit reduced histopathology, reduced organ proteinuria, and lower creatine levels than control rats. Using the three GNET models (anti-thymocyte serum, Con A -con A, and manual hypermassitis), Couser et al. Used recombinant sCR1 protein to determine the potential therapeutic efficacy of complement inhibition methods (Couser, WG, et al., J. Am. Soc. Nephrol 5: 1888-94, 1995). sRT1 treated rats exhibited significantly reduced PMN, platelet and macrophage inflow, reduced mesangial dissolution, and proteinuria compared to control rats. Further evidence for the importance of complement activation in glomerulonephritis was provided by the use of anti-C5 MoAb in the NZB / W Fl mouse model. Anti-C5 MoAbs inhibit the cleavage of C5, thus blocking the production of C5a and C5b-9. Continuous treatment with anti-C5 MoAb for 6 months resulted in a marked improvement in the symptoms of glomerulonephritis. A human adaptive anti-C5 MoAb monoclonal antibody (5G1.1), which prevents human complement component C5 from splitting into its pre-inflammatory component, is available from Alexion Pharmaceuticals, Inc. (New Haven, Connecticut) as a potential treatment for glomerulonephritis It is under development.

Direct evidence of the pathologic role of complement in renal damage was provided by the study of patients with a genetic deficiency of a specific complement component. Numerous studies have documented the relationship between kidney disease and the deficiency of complement regulator H (AuIt, BH, Nephrol. 74: 1045-1053, 2000; Levy, M., et al, Kidney Int. 30: 949-56, 1986; Pickering, MC, et al., Nat Genet., 31: 424-8, 2002). Factor H deficiency is manifested by low plasma levels of Factor B and C3 and consumption of C5b-9. Both atypical proliferative glomerulonephritis (MPGN) and idiopathic hemolytic uremic syndrome (HUS) are associated with factor H deficiency. Factor H deficient pigs (Jansen, JH, et al., Kidney Int. 53: 331-49, 1998) and factor H knockout mice (Pickering, M. C, 2002) - exhibit similar symptoms. Other complement component deficiencies are associated with renal disease and secondary to the development of systemic lupus erythematosus (SLE) (Walport, MJ, Davies, et al., Ann. NY Acad. ScL 815: 261-81, 1997 ). The absence of C1q, C4 and C2 makes SLE development much easier through mechanisms involving defective removal of immune complexes and apoptotic materials. In a number of such SLE patients, the onset of lupus nephritis, characterized by adherence of immune complexes through the glomeruli, occurs.

Further evidence for the link between complement activation and renal disease is provided by identification of autoantibodies to complement components in the patient, some of which are related to renal disease (Trouw, LA, et al., Mol. Immunol. 38: 199-206, 2001). Many such autoantibodies show that the term nephritic factor (NeF) is highly correlated with the kidney disease by which this activity is expressed. In clinical studies, approximately 50% of patients positive for nephritic factors developed MPGN (Spitzer, R.E., et al., Clin. Immunol. Immunopathol 64: 177-83, 1992). C3NeF is an autoantibody to the alternative pathway C3 converting enzyme (C3bBb), which stabilizes this converting enzyme and promotes alternative pathway activation (See, MR, et al., J. Immunol. 116: 1-7, 1976 ). Thus, an autoantibody that is specific for the classical pathway C3 converting enzyme (C4b2a), so-called C4NeF, stabilizes this converting enzyme and thus promotes classical pathway activation (see also MR et al., J. Immunol. 125 : 2051-2054, 1980; Halbwachs, L., et al., J. Clin. Invest., 65: 1249-56, 1980). Anti-C1q autoantibodies have been associated with nephritis in SLE patients (Hovath, L., et al., Clin. Exp. Rheumatol 19: 667-72, 2001; Siegert, C, et al., J. Rheumatol. 10: 19-23, 1992), the increase in the potency of this anti-C1q autoantibody is reported and predicts nephritic eruption (Siegert, C, et al., Clin. Exp. Rheumatol. Immune deposits eluted in post-kidney kidneys of SLE patients indicate accumulation of these anti-C1q autoantibodies (Mannick, M, et al., Am. J. Kidney Dis. All of these facts demonstrate the role of these autoantibody pathologies, but all patients with anti-C1q autoantibodies develop kidney disease (Siegert, CE, et al., Clin. Immunol. Immunopathol. 67: 204-9, 1993), and some healthy individuals also have low titers of anti-C1q autoantibodies.

Along with the alternative and classical pathway of complement activation, the lectin pathway may play an important pathological role in renal disease. Increased levels of MBL, MBL-associated serine proteases and complement activation products have been implicated in several other renal diseases such as Henoch-Schlenz spinal nephritis (Endo, M., et al., Am. J. Kidney Dis. 35: 401 Immunol. 101: 59-66, 2001) and IgA neuropathy (Endo, M., et al., Clin. Nephrology 55: 185-191, 2001) were detected by immunohistochemistry. Thus, although the link between complement and kidney disease has been known for decades, data on how complement affects precisely these kidney diseases is not complete.

One aspect of the present invention thus provides a method of treating a disorder or condition selected from the group consisting of glomerulonephritic glomerulonephritis, glomerulonephritis, < RTI ID = 0.0 > glomerulonephritis < / RTI > For the treatment of kidney symptoms including, but not limited to, glomerular capillary glomerulonephritis), glomerulonephritis after acute infection (glomerulonephritis after a streptococcal infection), cold globulin glomerulonephritis, lupus nephritis, Henoch-Schlenz spinal nephritis or IgA nephropathy . MASP-2 inhibitors may be administered to a subject by systemic administration, for example, by the administration of an artery, vein, intramuscular, subcutaneous or other parenteral administration, or potentially non-peptide. MASP-2 inhibitors may be administered periodically with extended time intervals to modulate or treat chronic conditions and may be administered once or repeated before, during, and / or after an acute trauma or injury.

Skin disorder

Psoriasis is a chronic, debilitating skin condition in which millions of people are affected and is affected by genetic and environmental factors. Topical and UVB and PUVA phototherapy are generally considered to be a frontline treatment of psoriasis. However, generalized or extended disease is used to strengthen UVB and PUVA therapy, either as primary therapy or, in some cases, systemic therapy.

The main pathogenesis of various skin disorders, such as psoriasis, supports the role of immune and proinflammatory processes, including the involvement of complement systems. Moreover, the role of the complement system has been established as an important nonspecific skin defense mechanism. Their activation not only helps maintain normal host defense but also induces the production of products mediating inflammation and tissue damage. The complement proinflammatory products of the complement include giant fragments of C3 with opsonin and cell-stimulating activities (C3b and C3bi), low molecular weight anapylatoxins (C3a, C4a, and C5a), and membrane attack complexes. Among them, C5a or its degradation product, C5a des Arg, appears to be the most important mediator because it exerts a strong chemotaxis on inflammatory cells. Intradermal administration of C5a anapylatoxin induces skin changes very similar to those observed in skin hypersensitivity vasculitis resulting from immune complex-mediated complement activation. Complement activation is implicated in the pathogenesis of inflammatory changes in autoimmune, vesicular dermatoses. Complement activation by pemphigus antibodies in the epidermis appears to be responsible for the development of eosinophilia, a change in lymphocyte inflammation. In the watery pemphigoid plexus (BP), the interaction of basement membrane antigen and BP antibody induces complement activation that appears to be involved in leukocyte inner dermal epidermal adhesion. Results Anaphylactosin activates invasive leukocytes and induces mast cell degranulation, which promotes dermal epidermal dissociation and eosinophil infiltration. Similarly, complement activation is likely to play a more direct role within the epidermis segregation, which is noted in acquired waterborne epidermolysis and gestational herpes.

Evidence of complement involvement in psoriasis can be found in the literature relating to the pathophysiological mechanisms of inflammatory changes in psoriasis and ductal disease, which have been recently tested. The growing body of evidence suggests that T-cell-mediated immunity plays an important role in triggering and maintaining psoriasis lesions. It has been shown that lymphocaine produced by activated T-cells in psoriatic lesions has a significant effect on epidermal proliferation. Characteristic neutrophil accumulation under the stratum corneum can be observed in areas of high inflammation of psoriatic lesions. Neutrophils are elicited by chemotaxis and are activated by C5a / C5a des-arg produced by chemokine synergistic activity, IL-8 and Gro-alpha release by stimulated keratinocytes, and alternative complement pathway activation (Terui , T., Tahoku J. Exp. Med., 190: 239-24%, 2000, Terui, T., Exp Dermatol, 9: 1-10, 2000).

Psoriasis scale extracts contain the only chemotactic peptide fractions that appear to be involved in the induction of rhythmic transdermal leukocyte chemotaxis. Recent studies have confirmed the presence of two unrelated chemotactic peptides in this fraction, namely C5a / C5a des Arg and interleukin 8 (IL-8) and their associated cytokines. Concentrations of immunoreactive C5a / C5a desArg and IL-8 in psoriatic lesion scale extracts and those associated with aseptic pustular dermatosis were quantitated to investigate the relative contribution of these correlations in transdermal leukocyte migration and psoriatic lesions. The concentrations of C5a / C5a desArg and IL-8 were found to be significantly increased in keratin-tissue extracts of lesional skin than those of non-inflamed normal keratinized skin. The increase in C5a / C5a desArg concentration was specific for lesion scale extracts. Based on these results, it appears that C5a / C5a desArg is produced only in inflammatory lesion skin under the specific environment favoring complement activation. This provides the rationale that psoriasis lesions are improved by the use of complement activation inhibitors.

Although the classic pathway of the complement system has been shown to be active in psoriasis, there is a report of the involvement of alternative pathways in the inflammatory response in small numbers of psoriasis. In the conventional view of the complement activation pathway, complementary fragments C4d and Bb are emitted at the time of classic and alternate path activation, respectively. The presence of the C4d or Bb fragment thus represents a progressive complement activation through the classical and / or alternative pathway. In one study, levels of C4d and Bb in psoriatic scale extracts were measured using enzyme immunoassay. The scale of these skin diseases contains higher levels of C4d and Bb detectable by enzyme immunoassay than those in the stratum corneum of noninflammatory skin (Takematsu, H., et al., Dermatologica 787: 289-292, 1990). These results indicate that alternative pathways are activated along with the classical pathways of complement in the psoriatic lesion skin.

Additional evidence for involvement of complement in psoriasis and atopic dermatitis was obtained by measuring normal complement components and activation products in the peripheral blood of 35 patients with atopic dermatitis (AD) in mild to moderate stages and 24 patients with psoriasis. Levels of C3, C4, and C1 deactivators (C1 INA) were measured in serum by radial immunodiffusion, while C3a and C5a levels were measured by radioimmunoassay. Compared with healthy non-atopic control, levels of C3, C4 and C1 INA were found to be significantly increased in both diseases. In AD, C3a levels tend to increase, whereas in psoriasis, C3a levels increase significantly. This result indicates that, in both AD and psoriasis, the complement system participates in the inflammatory process (Ohkonohchi, K., et al, Dermatologica 179: 30-34, 1989). Complement activation in the psoriatic lesion skin also results in attachment of the terminal complement complex in the epidermis defined by measuring the level of SC5b-9 in plasma and keratinocytes of psoriatic patients. The level of SC5b-9 in psoriasis plasma is significantly higher than that of control or atopic dermatitis patients. Studies on total protein extracts of lesional skin show that SC5b-9 levels are high in the lesional keratinocyte of psoriasis, although SC5b-9 was not detected in non-inflammatory keratinocytes. Immunofluorescence assay using monoclonal antibody against C5b-9 Neo antigen showed that the adhesion of C5b-9 was observed only in the stratum corneum of psoriasis skin. In summary, within the psoriatic lesion skin, the complement system is activated, and the complement activation proceeds all the way to the final stage of creating a membrane-attack complex.

New biological drugs that selectively target the immune system have recently been used in the treatment of psoriasis. Four biological drugs currently under FDA accreditation or in Phase 3 studies are: T-cell modulators alefacept (Amevive®) and efalizuMoAb (Raptiva®); etanercept (Enbrel), soluble TNF-receptor; And inflixiMoAb (Remicade®), an anti-TNF monoclonal antibody. Raptiva is an immune response modifier, in which the targeted mechanism of action is the blockade of the interaction between LFA-1 on lymphocytes and ICAM-1 on antigen-presenting cells and vascular endothelial cells. Binding of CD11a by Raptiva represents saturation of available CD11a binding sites on lymphocytes and down-change of cell surface CD11a expression on lymphocytes. This mechanism of action inhibits T-cell activation, dermal and epidermal cell migration, and T-cell re-activation. Thus, a number of scientific evidences point to the role of complement in inflammatory disease states of the skin, and recent pharmacological approaches have targeted immune systems or specific inflammatory processes. However, none of them identified MASP-2 as a targeted approach. Based on the inventor's new understanding of the role of MASP-2 in complement activation, the inventors believe that MASP-2 can be an effective target for the treatment of psoriasis and other skin disorders.

A feature of the present invention is therefore the treatment of psoriasis, autoimmune aberrant dermatosis, eosinophil-enhanced cotton, bullous pemphigoid, acquired papillary epidermolysis, atopic dermatitis, gynecological herpes and other skin disorders and induced capillary leakage The present invention relates to the administration of a composition comprising a therapeutically effective amount of a MASP-2 inhibitor in a pharmaceutical carrier to a subject having such a skin disorder. MASP-2 inhibitors can be administered topically or systemically, for example, by the application of a wash solution containing a spray, lotion, gel, paste, ointment or MASP-2 inhibitor such as, for example, intravenous, intramuscular, subcutaneous or other parenteral administration, Or potentially by non-peptidic inhibitors. The treatment may be a single dose or a repeated application or medication for acute symptoms, or a periodic application or medication for the control of chronic symptoms.

transplantation

Activation of the complement system contributes significantly to the inflammatory response after solid organ transplantation. In other recipients, the complement system is activated by ischemia / reperfusion, if possible, by antibodies to the graft (Baldwin, W. M., et al., Springer Seminol Immunopathol. 25: 181-197, 2003). In heterologous organ transplantation to non-primate exon primates, the main activator of complement is the preexisting antibody. Studies in animal models have shown that the use of complement inhibitors can significantly prolong graft survival (see below). Thus, there is an established role for the complement system in organ damage after organ transplantation, and thus the inventors have found that the use of complement inhibitors for MASP-2 can prevent damage to the grafts after homologous or heterologous transplantation.

Congenital immune mechanisms, especially complement, play a critical role in the inflammation and immune response to previously recognized grafts. For example, alternative complement pathway activation seems to mediate renal ischemia / reperfusion injury, and the body tubular cells may be both the source and the attack site of the complement component in this environment. The locally produced complement in the kidneys plays a role in developing both cellular and antibody-mediated immune responses to the graft.

C4d is the degradation product of the activated complement factor C4 which is a component of the classical and lectin-dependent pathway. C4d staining has emerged as a useful marker of humoral rejection in acute and chronic environments and has attracted new attention to the importance of anti-donor antibody formation. The relationship between C4d and morphological signals of acute cellular rejection is statistically significant. C4d is found in 24-43% Type I episodes, 45% Type II rejection and 50% Type III rejection (Nikeleit, V., et al, J. Am. Soc. Nephrol. 251, 2002, Nikeleit, V., et al., Nephrol Dial. Transplant 78: 2232-2239, 2003). Several therapies have been developed to reduce complement inhibition or local synthesis as a means to improve post-transplant clinical outcome.

Activation of the complement cascade occurs as a result of multiple processes during the implant. Current therapies, although effective in limiting cell-mediated rejection, do not address all of the obstacles faced. These include humoral rejection and chronic allograft nephropathy or dysfunction. Although the overall response to the transplanted organ is the result of a number of effector mechanisms in some parts of the host, complement may play a major role in some of these. In the context of kidney transplantation, complement local synthesis by the body tubular cells appears to be of particular importance.

The availability of complement specific inhibitors provides an opportunity for improved clinical outcome after organ transplantation. Inhibitors that act by mechanisms that block complement attacks are particularly useful because they have a promise for increased efficacy in already immunoprecipitated recipients and avoidance of systemic complement depletion.

Complement plays a crucial role in xenograft rejection. Thus, effective complement inhibitors are of great interest as potential therapeutic agents. In pig-primate organ transplantation, hyperacute rejection (HAR) is due to antibody attachment and complement activation. Multiple strategies and targets have been tested to prevent hyperplastic rejection in pig-primate combinations. This approach is accompanied by removal of natural antibodies, complement depletion by cobra venom factor, or prevention of C3 activation by the soluble complement inhibitor sCR1. In addition, recombinant soluble chimeric proteins derived from complement activation blocker-2 (CAB-2), human destructive acceleration factor (DAF) and membrane cofactor proteins inhibit both the classical and alternative pathway C3 and C5 converting enzymes. CAB-2 reduces complement-mediated tissue damage of pig hearts inoculated with human blood in vitro. Studies of CAB-2 efficacy have shown that graft survival was record- edly prolonged in CAB-2 transplanted monkeys when pig hearts were transplanted positively into a monkey that was not immunosuppressed (Salerno, CT, et al, Xenotransplantation 9: 125-134, 2002). CAB-2 has been shown to inhibit complement activation in vivo and is shown to be strongly reduced by the production of C3a and SC5b-9. In graft rejection, tissue attachment of iC3b, C4 and C9 was similar or slightly decreased compared to the control, and IgG, IgM, C1q and fibrin attachment did not change. Thus, this approach to complement inhibition eliminated the hyperacute rejection of swine hearts implanted in a monkey. These studies demonstrate the advantages of complement inhibitory effects on survival and we believe that MASP-2 inhibition may be useful for xenotransplantation.

Another approach is to determine whether it is possible to prevent hyperacute rejection (HAR) in a rat-senescent-sensitized mouse heart transplant model and whether such MoAbs coupled to cyclosporin and cyclosporamid can achieve organ graft survival And the like. We have found that anti-C5 MoAb prevents HAR (Wang, H., et al., Transplant (58: 1643-1651, 1999). The present inventors have therefore found that other targets in the complement cascade, such as MASP- It is believed that it may be worthwhile to prevent HAR and acute vascular rejection in xenogeneic organ transplants.

The main role of complement in the hyperacute rejection observed in xenografts is well established, but has a minor role in allogeneic transplantation. The link between complement and acquired immune response is known, along with the fact that complement-depleted animals show an abnormal antibody response after antigen challenge. The opsonization of the antigen by the complement cleavage product C3d has been shown to significantly increase the antigen presentation effect on B cells and appears to be exerted through the involvement of complement receptor 2 on certain B cells. This study can be extended to the implantation environment of the mouse skin graft model where C3- and C4-deficient mice have significant defects in homolog-antibody production, which leads to failure of class switching to high-affinity IgG . The importance of this mechanism in renal transplantation is increased, which is due to the importance of anti-donor antibodies and humoral rejection.

Previous studies have already demonstrated the upregulation of C3 synthesis by endothelium-derived cells in allogeneic graft rejection after renal transplantation. The role of local synthetic complement was investigated in the mouse kidney transplantation model. The C3-negative donor transplantation into the C3-full recipient proved that the survival time was prolonged (> 100 days) compared to the C3-positive donor transplantation control, which resulted in rejection within 14 days. Additionally, the anti-donor T-cell proliferative response in the recipient of C3-negative grafts was record- edly reduced compared to that of the control, indicating the effect of locally synthesized C3 on T-cell priming.

These observations suggest that the donor antigen may be the first to develop in the graft by exposure to T-cells and that the locally synthesized complement may be mediated by opsonization of donor antigens or by additional signaling to both antigen-presenting cells and T- Suggesting the possibility of enhancing antigen presentation. Under this kidney transplantation environment, complement production tubular cells demonstrate complement attachment on the cell surface.

One aspect of the present invention thus provides a method of treating a subject comprising administering to a pharmaceutical carrier a composition comprising a therapeutically effective amount of a MASP-2 inhibitor in a pharmaceutical composition comprising a compound of formula (I), or a pharmaceutically acceptable salt, solvate or prodrug thereof, , Bone marrow, etc.), or a transplant recipient comprising a recipient with a heterologous organ transplant, to prevent or treat an inflammatory response due to tissue or solid organ transplantation. The MASP-2 inhibitor may be administered to a subject by arterial, intravenous, intramuscular, subcutaneous or other parenteral administration, or by oral administration of a potentially non-peptidic inhibitor. Administration can occur as an acute phase after transplantation and / or as a therapy after organ transplantation. In addition to or instead of post-transplant administration, the subject may be treated with a MASP-2 inhibitor prior to and / or during the transplantation procedure, and / or may be treated by pre-treatment with a MASP-2 inhibitor to the transplanted organ or tissue. Organs or tissues may be prepared by spraying a solution, a gel or paste containing a MASP-2 inhibitor on the surface of organs or tissues, or by washing the surface, or by immersing organs or tissues in a solution containing the MASP-2 inhibitor .

Central and peripheral nervous system disorders and damage

Activation of the complement system may be used to treat multiple sclerosis (MS), myasthenia gravis (MG), Huntington's chorea (HD), amyotrophic lateral sclerosis (ALS), acute idiopathic neuropathies, post-stroke reperfusion, degenerative disc, (CNS) or peripheral nervous system (PNS) disease or injury, including, but not limited to, Alzheimer ' s disease (AD) and Alzheimer ' s disease (AD). The initial determination that complement proteins are synthesized in CNS cells, including neurons, astrocytes and macrophages, and the awareness that anapylatoxin generated in the CNS, by complement activation, (Morgan, BP, et al., Immunology Today 17: 10: 461-466, 1996). C3a and C5a receptors have been found on neurons and have been found to be widely distributed in sensory, motor and specific regions of the brain marrow system (Barum, SR, Immunologic Research 2 (5: 7-13, 2002) C5a and C3a have been shown to alter dietary habits in rodents and can induce calcium signaling in microglial cells and neurons This finding is associated with a variety of CNS inflammatory diseases including brain trauma, dehydration, meningitis, stroke and Alzheimer's disease And develops possibilities for therapeutic agents that inhibit the activation of mast cells.

Brain trauma or hemorrhage is a common clinical problem, and complement activation can occur, which can aggravate inflammation and edema. The effect of complement inhibition has been studied within the brain trafficking model (Kaczorowski et al., J. Cereb. Blood Flow Metab. 75: 860-864, 1995). Administration of sCR1 significantly inhibits neutrophil infiltration into the injured site just prior to brain injury, indicating that complement is important for the use of phagocytic cells. Thus, complement activation after cerebral hemorrhage is clearly influenced by the presence of high levels of multiple complement activation products in both plasma and cerebrospinal fluid (CSF). Complement activation and increased staining of the C5b-9 complex has been demonstrated in isolated spinal disc tissues and may suggest a role in disk healing tissue-induced sciatica (Gronblad, M., et al., Spine 28 2): 114-118, 2003).

MS is characterized by a radical reduction in myelin that encapsulates and insulates axons within the CNS. Although the cause of the initiation is unknown, there is a large amount of evidence that the immune system is involved (Prineas, JW, et al., Lab Invest. 38: 409-421, 1978; Ryberg, B., J. Neurol. ScL 54: 239-261, 1982). There is clear evidence that complement has a distinct role for the pathophysiology of CNS or PNS dehydration diseases, including MS, Guillain-Barre syndrome and Miller-Fisher syndrome (Gasque, P., et al., Immunopharmacology 49: 171-186 , 2000; Barnum, SR in Bondy S. et al. (Eds.) Inflammatory events in neurodegeneration, Prominent Press 139-156, 2001). Complement contributes to tissue destruction, inflammation, removal of the myelin debris, and even rejuvenation of the axon. Despite the obvious evidence of complement involvement, confirmation of complement therapy targets was currently only assessed within animal models of experimental allergic encephalomyelitis (EAE), multiple sclerosis. Studies have established that EAE mouse deficiency in C3 or factor B attenuated dehydration kinship compared to EAE control mice (Barnum, Immunologic Research 2 (5: 7-13, 2002). The soluble form of the complement inhibitor "sCrry" EAE mouse studies using C37 - / - and factor B - / - have demonstrated that complement contributes to the development and progression of the disease model at various levels. In addition, a significant reduction in the EAE severity in the factor B - / - Provides additional evidence of an alternative pathway of complement in the EAE (Nataf et al., J. Immunology 765: 5867-5873, 2000).

MG is a neuromuscular connective disease with loss of acetylcholine receptors and destruction of the terminal plate. sCR1 is highly effective in MG animal models, which further indicate the role of complement in disease (Piddelesden et al., J. Neuroimmunol. 1997).

Neurodegenerative diseases, which are histologic hallmarks of AD, are senescent plaque and nerve fiber thickeners (McGeer et al., Res. Immunol. 143: 621-630, 1992). These pathological markers are a powerful source of complement system components. Evidence suggests local neuroinflammatory conditions leading to nerve death and cognitive impairment. Senile plaques palm oil amorphous amyloid-D peptide (AD, peptide derived from amyloid precursor protein). AD binds to C1 and triggers complement activation (Rogers et al., Res. Immunol. 143: 624-630, 1992). In addition, the hallmark of AD is the binding of the activated protein of the C1q-C5b-9 classical complement pathway, which is found to be highly localized in neurogenic plaques (Shen, Y., et al., Brain Research 769: 391- 395, 1997; Shen, Y., et al., Neurosci. Letters 305 (3): 165- 168, 2001). Thus, not only is AD initiated in the classical pathway, but the resulting persistent inflammatory state may also contribute to neuronal cell death. Moreover, the fact that it proceeds to the complement activation constant C5b-9 in AD indicates that the regulatory mechanism of the complement system does not stop the complement activation process.

Several complement pathway inhibitors have been proposed as potential therapeutic methods for AD, including C1q binding inhibitor proteoglycans, C3 converting enzyme inhibitor Nafamstat, and C5 activating blockers or C5a receptor inhibitors (Shen, Y., et al. Progress in Neurobiology 70: 463-472, 2003). As a start-up step in the innate complement pathway, and the role of MASP-2 for alternative pathway activation, it provides a potential new therapeutic approach and is supported by the health of the data suggesting complement pathway involvement in AD.

There is evidence of inflammation characterized by increased expression of glial cells (especially small cell) responses, and HLA-DR antigens, cytokines, and complement components, in the area of brain injury in PD patients as other CNS degenerative diseases. These observations imply that they are involved in the pathogenesis of nerve damage in the immune system mechanism PD. The cellular mechanism of primary injury in PD has not been classified yet. However, mitochondrial mutations, oxidative stress and apoptosis appear to play a role. In addition, inflammation initiated by nerve damage of the striatum and substantia nigra in the PD worsens the condition of the disease. These observations indicate that treatment of complement inhibitory drugs acts to slow PD progression (Czlonkowska, A., et al., Med. ScL Monit. 8: 165-177, 2002).

(PNS) and / or central nervous system (CNS) disorders by administering to a subject having such disorder or disorder a composition comprising a therapeutically effective amount of a MASP-2 inhibitor in a pharmaceutical carrier, Or < / RTI > CNS and PNS disorders and impairments that can be treated according to the present invention are selected from the group consisting of multiple sclerosis (MS), severe myasthenia gravis (MG), Huntington's chorea (HD), amyotrophic lateral sclerosis (ALS), acute idiopathic multiple sclerosis, Including, but not limited to, inflammatory bowel disease, diabetic retinopathy, dyspnea, brain trauma, PD, Alzheimer's disease, Miller-Fisher syndrome, brain trauma and / or hemorrhage, dehydration and, if possible, meningitis.

To treat CNS symptoms and brain trauma, MASP-2 inhibitors may be administered orally, intrathecally, intracranially, intracerebroventricularly, intraarterially, intravenously, intramuscularly, subcutaneously, or other parenterally, and potentially non-peptide inhibitors And the like. PNS symptoms and brain trauma can be treated by systemic administration or alternatively by local administration of dysfunctional or traumatic areas. Administration of the MASP-2 inhibiting composition of the present invention may be repeated periodically by the clinician until effective control or symptom control is achieved.

Blood disorder

Sepsis is caused by a patient's overwhelming response to microbial invasion. The main function of the complement system is to harmonize the inflammatory response to invading bacteria and other pathogens. Consistent with this physiological role, complement activation has been shown to play a major role in the pathogenesis of sepsis in a number of studies (Bone, R. C., Annals. Internal. Med. 775: 457-469, 1991). The definition of clinical signs of sepsis is ongoing. Sepsis is generally defined as a systemic host response to an infection. However, in many cases, clinical evidence of infection (e.g., positive bacterial blood cultures) has not been found in patients with infection symptoms. This discrepancy has been established and considered as the term "systemic inflammatory response syndrome" (SIRS) at the 1992 consensus conference and does not require a definitive presence of bacterial infection (Bone, RC, et al., Crit. : 724-726, 1992). There was general consensus that sepsis and SIRS were accompanied by an inability to control the inflammatory response. The purpose of this brief review is to include severe septicemia, septic shock, and SIRS in the clinical definition of sepsis.

Prior to the late 1980s, the main source of infection in infected patients was Gram-negative bacteria. Lipopolysaccharide (LPS), a major component of gram-negative bacterial cell walls, is known to stimulate the release of inflammatory mediators in a variety of cell types and to induce acute infectious symptoms upon injection into animals (Haeney, MR, et al. , Antimicrobial Chemotherapy 47 (Suppl. A): 41-6, 1998). Interestingly, the spectrum of causative microorganisms seems to have migrated from gram-negative bacteria prevailing in the late 1970s and late 1980s to current predominant Gram-positive bacteria, and the reason is still unclear (Martin, GS, et al. Eng. J. Med., 348: 1546-54, 2003).

Numerous studies have shown the importance of complement activation in inflammatory mediators and are known to contribute to the characteristics of shock, particularly infection and hemorrhagic shock. Both gram-negative and gram-positive organisms are generally involved in septic shock. LPS is a potent activator of complement through an alternative pathway, although it may occur by classical pathway activation mediated by the antibody (Fearon, DT, et al., N. Engl. J. Med. 292: 937-400, 1975 ). The major components of Gram-positive cell walls are peptidoglycan and lipidic tyrosic acid, and although they can activate the classical complement pathway in the presence of specific antibodies, the two components are potential activators of the alternative complement pathway (Joiner, KA, et al., Rev. Immunol. 2: 461-2, 1984).

The complement system is involved early in the pathogenesis of septicemia because of the attention of researchers that anapylatoxins C3a and C5a mediate the various inflammatory responses that may occur during septicemia. These anapylatoxins cause vasodilatation, increased microvascular permeability, and central symptoms of septic shock (Schumacher, W.A., et al., Agents Actions 34: 345-349, 1991). In addition, anapylatoxin includes bronchospasm, histamine release from mast cells, and platelet aggregation. Moreover, they exert a number of effects on the coagulation, coagulation, adhesion, release, production of toxic peroxidic anions and formation of leukotrienes of granulocytes such as lysosomal enzymes (Shin, HS, et al., Science 162: 361-363, 1968 ; Vogt, W., Complement 3: 177-86, 1986). These biological effects are known to play a role in the development of sepsis complications such as shock or acute respiratory distress syndrome (ARDS) (Hammerschmidt, DE, et al., Lancet 7: 947-949, 1980; Slotman, GT, , Surgery 99: 744-50, 1986). In addition, increased levels of anaphylactosin C3a have been associated with the fatal outcome of sepsis (Hack, C. E., et al., Am. J. Med. 86: 20-26, 1989). In some shock animal models, certain complement-deficient strains (e.g., C5-deficient) are more resistant to the effects of LPS infusion (Hseuh, W., et al., Immunol. 70: 309-14, 1990). In the case of sepsis in rodents, the blockade of C5a production by antibodies has a significantly increased survival (Czermak, B. J., et al., Nat. Med. 5: 788-792, 1999). A similar finding appears in antibodies or small molecule inhibitors when the C5a receptor (C5aR) is blocked (Huber-Lang, MS, et al., FASEB J. 16: 1561-14, 2002; Riedemann, NC et al., J Clin. Invest. 770: 101-8, 2002). Early monkey experimental studies showed that antibody blockade of C5a resulted in an increase Inducing septic shock and adult respiratory distress syndrome (Han, G., et al., J. Surg. Res. 46: 195-9, 1989; Stevens, JH, et al., J Clin. Invest. 77: 1812-16, 1986). In humans with septicemia, C5a is increased and survival rate is significantly reduced with multi-organ shutdowns compared to less severely infected patients and survivors (Nakae, H., et al., Res. Commun. Chem. Pathol Bengtson, A., et al., Arch. Surg., 123: 645-649, 1988). The mechanism by which C5a exerts its adverse effects on sepsis has not yet been elucidated, and recent data suggest that C5a production during septicemia is associated with the innate immune function of blood neutrophils (Huber-Lang, MS, et al., J. Immunol. 169: 3223- (Riedamann, NC, et al., Immunity 79: 193-202, 2003), their ability to express respiratory exacerbations, and their ability to produce cytokines. In addition, C5a production during septicemia appears to have a coagulation effect (Laudes, L., et al., Am. J. Pathol. 160: 1867-75, 2002). The complement-regulatory protein CI INH has efficacy in animal models of sepsis and ARDS (Dickneite, G., Behring Ins. Mitt. 93: 299-305, 1993).

The lectin pathway plays a role in the pathogenesis of sepsis. MBL activates the lectin pathway and binds a range of clinically important microorganisms including both gram-negative and gram-positive bacteria (Neth, O., et al., Infect. Immun. , 2000. Lipidic tycoic acid (LTA) is considered a gram-positive counterpart of LPS, a potent immunostimulant that induces cytokine release in mononuclear cells and whole blood (Morath, S. et al. Recently, L-picolin has been shown to be effective against a number of Gram-positive bacterial species, such as Staphylococcus aureus (Staphylococcus aureus, Streptococcus aureus, Streptococcus aureus, (Lynch, NJ, et al., J. Immunol. 772: 1198-02, 2004) MBL is a protein that specifically binds to Enterococcus spp And the polyglycerophosphate chain is known to bind to the LTA of the glycosyl group (Polotsky, V. Y., et al., Infect. Immun. (54: 380, 1996)).

A feature of the present invention is therefore to provide a method of treating a subject suffering from the symptoms of sepsis or sepsis, including but not limited to severe sepsis, septic shock, acute respiratory distress syndrome due to sepsis, and systemic inflammatory response syndrome, The present invention provides a method of treating sepsis or sepsis symptoms by administering a composition comprising a therapeutically effective amount of an inhibitor. A related method includes administering to a subject suffering from such a condition a composition comprising a therapeutically effective amount of a MASP-2 inhibitor in a pharmaceutical carrier to treat hemorrhagic shock, hemolytic anemia, autoimmune thrombotic hemolytic anemia (TTP), hemolytic uremic syndrome (HUS) And other blood disorders including other bone marrow / blood-clotting symptoms. MASP-2 inhibitors may be administered to a subject by systemic administration, for example, by the arterial, intravenous, intramuscular, inhalation (especially ARDS), subcutaneous or other parenteral administration, or potentially non-peptide. The MASP-2 inhibitor composition may be combined with one or more additional therapeutic agents to counter the onset of sepsis and / or shock. For advanced sepsis or shock or distress symptoms caused by them, the MASP-2 inhibitory composition is administered in a rapid-active dosage form, for example, by intravenous or arterial delivery of bolus of a solution containing the MASP-2 inhibitor composition It is appropriate. Repeat dosing may be performed according to the clinician's decision until the symptoms are resolved.

Urinary Reproductive Symptoms

The complement system may be used to treat pain, bladder disease, sensory bladder disease, chronic aseptic cystitis, and interstitial cystitis (Holm-Bentzen, M., et al., J. Urol. 138: 503-501, Fetal maternal resistance (Xu, C, et al., Science 287: 498-507, 2000), pregnancy (Xu, C., et al, Science 287: 498-507, 2000), Biol. Reprod. 498-507, 2000) and pre-infarction angina (Haeger, M., Int. J. Gynecol. Obstet. 43: 113-121, 1993).

Pain bladder disease, sensory bladder disease, chronic aseptic cystitis, and interstitial cystitis are undefined symptoms of unknown etiology and pathogenesis, and therefore, they have no rational treatment. Pathological theories related to defects in the epithelium and / or mucosal surface coating of the bladder, and immunological disturbance predominate (Holm-Bentzen, M., et al., J. Urol. 138: 503-501, 1987 ). It has been reported that interstitial cystitis patients have been tested for immunoglobulins (IgA, G, M), complement components (C1q, C3, C4) and C1-esterase inhibitors. There is a very pronounced depletion of serum levels of complement component C4 (p 0.001 or less) and immunoglobulin G significantly increased (p 0.001 or less). This study demonstrates the activation of the classical pathway complement system and suggests that chronic local immunological processes are involved in the pathogenesis of this disease (Mattila, J., et al., Eur. Urol. 9: 350-352, 1983 ). Furthermore, following binding of autoantibodies to antigens in bladder mucosa, complement activation may be involved in the production of tissue damage typical to this disease and chronic self-sustaining inflammation (Helin, H., et al., Clin. Immunol. Immunopathol., 43: 88-96, 1987). Together with the role of complement in urogenital inflammatory disease, the reproductive function may be affected by local regulation of the complement pathway. Naturally occurring complement inhibitors have been implicated in providing protection to the host cells in which they need to regulate the body's complement system. Crry, a naturally occurring rodent complement inhibitor that is structurally similar to human complement inhibitors, MCPs and DAFs, has been investigated to describe controlled controls of complement in fetal development. Interestingly, attempts to generate Crry - / - mice were successful. Instead, it was found that homozygous Crry - / - mice died within the uterus. Crry - / - embryos survived until 10 days after sexual intercourse, and survival was rapidly reduced due to developmental arrest. Inflammatory cells invaded into placental tissues of Crry - / - embryos. In contrast, the Crry + / + embryo appears to retain C3 attached to the placenta. This indicates that complement activation occurred at the placental level, in the absence of complement control, and that the embryo died. Confirmatory studies investigated the introduction of Crry mutations on the C3 deficiency background. This structural strategy was successful. At the same time, this data indicates that the fetal maternal complement interface must be regulated. Minor changes in complement regulation in the placenta contribute to placental dysfunction and abortion (Xu, C, et al., Science 287: 498-507, 2000).

Pre-infarction angina is a pregnancy-induced hypertensive disorder in which complement system activation is involved but remains controversial (Haeger, M., Int. J. Gynecol. Obstet., 43: 113-127, 1993). Complement activation in the systemic circulation is closely related to established disease in preinfarct angina and does not show any increase before the presence of clinical symptoms. Therefore, complement components can not be used as predictors of preinfarct angina (Haeger, et al., Obstet. Gynecol. 78: 46,1991). However, increased complement activation in the local environment of the placenta bed can overcome local control mechanisms and exhibit increased levels of anapylatoxin and C5b-9 (Haeger, et al, Obstet. Gynecol. 73: 551, 1989 ).

The infertility mechanism associated with the proposed single antitactor antibody (ASA) is through the role of complement activation in the reproductive tract. The production of C3b and iC3b opsonin which enhance sperm binding by phagocytic cells through their complement receptors and the formation of a terminal C5b-9 complex on the sperm surface is a potential cause of decreased pregnancy rate. Increased levels of C5b-9 have been demonstrated in ovarian follicular fluid of infertile women (D'Cruz, OJ., Et al., J. Immunol. 744: 3841-3848, 1990). Other studies show impaired sperm motility and reduced sperm / egg interaction, which is complement dependent (D'Cruz, OJ, et al., J. Immunol. 146: 611-620, 1991; Alexander, NJ, Fertil, Steril, 47: 433-439, 1984). Finally, studies on sCR1 have demonstrated protective effects on ASA- and complement mediated damage to human sperm (D'Cruz, O. J., et al., Biol. Reprod. 54: 1217-1228, 1996). These data provide some evidence for the use of complement inhibitors in the treatment of urogenital diseases and disorders.

One aspect of the invention thus provides a method of inhibiting MASP-2-dependent complement activation in a patient suffering from genitourinary disorders by administering to the subject suffering from such a disorder a composition comprising a therapeutically effective amount of a MASP-2 inhibitor in the pharmaceutical carrier . The urogenital disorders that can be treated with the compositions and methods of the present invention include, but are not limited to, pain bladder diseases, sensory bladder diseases, chronic aseptic cystitis and interstitial cystitis, male and female infertility, placental dysfunction and abortion and infarction I include angina. MASP-2 inhibitors may be administered to a subject by systemic administration, for example, by intravenous, intravenous, intramuscular, inhalation, subcutaneous or other parenteral administration, or potentially non-peptide. Alternatively, the MASP-2 inhibitory composition can be a local delivery to the genitourinary tract by, for example, perfusion in the bladder or insertion of a liquid solution or gel composition. Repeated dosing may be performed at the clinician's discretion to control or alleviate symptoms.

Diabetes and diabetes symptoms

Diabetic retinal microvascular disease is characterized by increased permeability, leukocyte congestion, microthrombosis, and apoptosis of capillary cells, all of which can be induced or promoted by complement activation. Diabetic glomerular structure and neuroendocrine microvessels show signs of complement activation. The reduced utility or effect of complement inhibitors in diabetes is the glycosylphosphatidylinositol (GPI) -anchored membrane protein on the endothelial cell surface by in vitro high glucose, and CD59 inhibits their complement-inhibitory function Indicating the selective reduction of the expression of two inhibitors, CD55 and CD59, which undergo non-enzymatic glycation. Zhang et al. (Diabetes 57: 3499-3504, 2002) investigated the involvement of complement activation and inhibitory molecules as a feature of human non-proliferative diabetic retinopathy. Adherence of C5b-9, the final product of complement activation, occurs in the retinal vasculature of human eye donors with type 2 diabetes, but not in the blood vessels of age-matched non-diabetic donors. The only complement components C1q and C4 in the classical pathway are not detected in the diabetic retina, indicating that C5b-9 was produced by the alternative pathway. Diabetic donors show a marked reduction in retinal levels of CD55 and CD59, and these two complement inhibitors are bound to the plasma membrane by GPI anchors. Similar enhancement of complement activation in the retinal vessels and selective reduction of levels of retinal CD55 and CD59 was observed in the 10 week transcript of streptozotocin-induced diabetes. Thus, diabetes appears to be due to a regulatory defect in complement activation and complement activation preceded by other signs of diabetic retinal microvascular disease.

Gerl et al. (Investigative Ophthalmology and Visual Science 43: 1104-08, 2000) determined the presence of activated complement components in the eyes affected by diabetic retinopathy. Immunohistochemistry studies have found extensive attachment of the complement C5b-9 complex, which is immediately accumulated in the bottom composite layer in all 50 diabetic retinopathy specimens and detected in the choroidal capillary layer thickly surrounding the capillaries. C3d staining correlates positively with C5b-9 staining, indicating that complement activation occurred in situ. In addition, positive staining is found in bitronectin, which forms a stable complex with extracellular C5b-9. In contrast, there is no positive staining for C-reactive protein (CRP), mannan-binding lectin (MBL), C1q, or C4, indicating that complement activation does not occur via the C4-dependent pathway. Thus, the presence of C3d, C5b-9, and bitronectin indicates that complement activation is achieved through an alternative pathway in the choroidal capillary layer of the eye, if possible, affected by diabetic retinopathy.

Complement activation may be a cause of pathological sequelae that may contribute to ocular tissue disease and visual impairment. Thus, the use of complement inhibitors can be an effective therapy to reduce or prevent microvascular injury that can occur in diabetes.

Insulin-dependent diabetes mellitus (IDDM, type-I diabetes) is an autoimmune disease associated with the presence of other types of autoantibodies (Nicoloff et al., Clin. Dev. Immunol. 11: 61-66, 2004). The presence of these antibodies and their corresponding antigens in the circulation induces the formation of the circulating immune complex (CIC), which is known to remain in the blood for a long period of time. Adherence of CICs in small blood vessels is capable of inducing microangiopathy that weakens the clinical outcome. There is a hypothetical relationship between the development of CIC and microvascular complications in diabetic children. This finding indicates that increased levels of CIC IgG are associated with early development of diabetic nephropathy and that inhibitors of complement pathway are effective in blocking diabetic nephropathy (Kotnik, et al., Croat. Med. J. 44: 707-11, 2003). In addition, the formation of downcomplement proteins and the involvement of alternative pathways appear to be contributors to the overall islet cell function in IDDM, and the use of complement inhibitors to reduce potential damage or limited cell death is expected (Caraher, et al., J. Endocrinol. 162: 143-53, 1999).

Circulating MBL concentration is significantly increased in type 1 diabetic patients compared to healthy controls, and this MBL concentration is positively correlated with urinary albumin excretion (Hansen et al., J. Clin. Endocrinol. Metab. 88: 4857-61 , 2003). Recent clinical studies have found that the frequencies of the high- and low-expression MBL genotypes are similar to those of type 1 diabetic patients (Hansen et al., Diabates 53: 1570-76, 2004). However, the risk of developing kidney disease among diabetic patients is significantly increased when they have a high MBL genotype. Suggesting that high MBL levels and lectin pathway complement activation may contribute to the development of diabetic nephropathy. This conclusion is supported by a recent prospective study in which a relationship between MBL levels and albuminuria was examined in a cohort study of newly diagnosed Type 1 diabetic patients (Hovind et al., Diabetes 54: 1523-21, 2005). They found that high levels of MBL in the early stages of type 1 diabetes were significantly associated with subsequent development of albuminuria. These results indicate that the MBL and lectin pathways are involved in the specific pathogenesis of diabetic complications rather than merely accelerating the changes present. In a recent clinical study (Hansen et al., Arch. Intern. Med. 166: 2007-13, 2006), MBL levels were measured at baseline within a well-characterized cohort study of type 2 diabetic patients followed for 15 years Respectively. They found that after adjustment for known disturbances, the risk of death was significantly higher among patients with high MBL plasma levels (> 1000 μg / L) than patients with low MBL levels (<1000 μg / L).

In another aspect, the present method includes administering a composition comprising a therapeutically effective amount of a MASP-2 inhibitor in a pharmaceutical carrier for the treatment of vascular disease of non-vital diabetes mellitus (IDDM) or IDDM or adult type (type 2) Dependent complement activation in a subject suffering from neuropathy or retinopathy complications. MASP-2 inhibitors may be administered to a subject by systemic administration, for example, by the administration of an artery, vein, intramuscular, subcutaneous or other parenteral administration, or potentially non-peptide. Alternatively, the administration can be administered locally to the site of vascular disease, neuropathy or retinopathy symptoms. MASP-2 inhibitors can be single or sequential doses for prolonged periods of time for the treatment or control of chronic conditions, or for the treatment of acute symptoms.

Post- chemotherapy and treatment of malignant tumors

Activation of the complement system may be involved in the pathogenesis of malignant tumors. Recently, newborn antigens of C5b-9 complement complex, IgG, C3, C4, S-protein / bitonectin, fibronectin, and macrophage were immunized with 6 positive The breast tumor samples and 17 breast cancer samples were localized. All tissue samples with carcinomas of each TNM stage presenting C5b-9 are attached to tumor cell membranes, thin granules on cell remnants, and diffusion attachments in the necrotic area (Niculescu, F., et al., Am. Pathol, 140: 1039-1043,1992).

In addition, since complement activation is the result of chemotherapy or radiotherapy, inhibition of complement activation is therefore useful as an adjunct in the treatment of malignant tumors to reduce prostate inflammation. When chemotherapy and radiation therapy precedes surgery, C5b-9 attachment becomes more intense and expanded. C5b-9 adhesion was absent in all benign lesions. The S-protein / bitronectin is provided as a fibrous attachment in the connective tissue matrix and as a diffusion attachment around the tumor cells that is less potent and less dilated than fibronectin. IgG, C3, and C4 adherence only appear in carcinoma samples. The presence of C5b-9 attachment is an index of complement activation and its subsequent pathological effect in breast cancer (Niculescu, F., et al., Am. J. Pathol. 140: 1039-1043, 1992).

Pulsed-adjustable dye laser (577 nm) (PTDL) therapy induces hemoglobin clotting and tissue necrosis, and is mainly restricted to blood vessels. In the PTDL-irradiated normal skin study, the major findings were as follows: 1) C3 fragment, C8, C9, and MAC attached to the vessel wall; 2) This attachment was not due to protein denaturation because 7 min after irradiation, unlike the immediate attachment of transferrin at the site of erythrocyte solids; 3) C3 attachment indicates amplification of complement activation by an alternative pathway, which is a specific response, since tissue necrosis itself did not induce amplification; And 4) this reaction promotes the local accumulation of polymorphonuclear leukocytes. Tissue necrosis is more pronounced in hemangiomas. The hemangioma vessels in the middle of necrosis do not fix complement significantly. In contrast, endovascular fixation at the perimeter is similar to that observed in normal skin with one exception: C8, C9, and MAC were detected immediately in the same vessel after laser treatment, and this finding was consistent with the formation of C5-converting enzyme This is consistent with the combination of direct MACs without These results indicate that complement is activated in PTDL-induced vascular necrosis and assures an inflammatory response.

Photodynamic therapy (PDT) of tumors induces a strong host immune response, one of which is pronounced neutrophilia. There are at least twelve secondary mediators that arise as a result of complement activation, with released complement fragments (direct mediator) released as a result of PDT-inducible complement activation. The latter includes cytokines IL-1 beta, TNF-alpha, IL-6, IL-10, G-CSF and KC, thromboxane, prostaglandins, leukotrienes, histamines and clotting factors (Cecic, L, et al. Cancer Lett. 785: 43-51, 2002).

Finally, the use of MASP-2-dependent complement activation inhibitors was planned in connection with standard therapeutic regimens of cancer therapy. For example, chimeric anti-CD20 monoclonal antibody, rituximab therapy, is associated with moderate to severe first-dose adverse effects, especially in patients with a high number of circulating tumor cells. Recent studies at the first infusion of rituximab have shown that complement activation products (C3b / c and C4b / c) and cytokines (tumor necrosis factor alpha (TNF-alpha), interleukin 6, IL-6 and IL-8 Injection of rituximab induces rapid complement activation and promotes the release of TNF-alpha, IL-6 and IL-8. The level of complement activation seems to correlate with the number of pre-injection B-cell circulations (r = 0.85; P = 0.07) and adverse event severity, indicating that it plays a central role in the pathogenesis of rituximab treatment adverse events. Since complement activation can not be prevented by corticosteroids, this may be related to the possible role of complement inhibitors in the initial administration of rituximab (van der Koik, LE, et al, Br. J. Haematol. 775: 807-811, 2001).

Another aspect of the present invention provides a method of inhibiting MASP-2-dependent complement activation in a subject who has received treatment with a chemotherapeutic agent and / or radiotherapy that inhibits MASP-2-dependent complement activation. The method comprises administering to the patient a composition comprising a therapeutically effective amount of a MASP-2 inhibitor in a pharmaceutical carrier before or after chemotherapeutic, i.e., chemotherapeutic agent (s) and / or administration of radiation therapy &Lt; / RTI &gt; For example, administration of an inventive MASP-2 inhibitor composition can be used to reduce the crucial effect of chemo- and / or radiotherapy within a non-targeted healthy tissue, simultaneously or prior to chemo- or radiotherapy, and during the course of therapy Can be carried out continuously. Together, the MASP-2 inhibitor composition may be administered after chemo-and / or radiation therapy. It should be understood that chemo-and radiotherapy requires repeated treatment, thus it is possible that administration of the MASP-2 inhibitor composition can be repeated in accordance with the chemotherapeutic agent and the light irradiation treatment. MASP-2 inhibitors may be used alone or in combination with other chemotherapeutic agents and / or radiotherapy as chemotherapeutic agents for the treatment of malignant tumor patients. Administration is preferably oral (non-peptidic), intravenous, intramuscular or via other parenteral routes.

Endocrine disorder

The complement system has recently been shown to be effective in the treatment of hypothyroidism (Blanchin, S., et al., Exp. Eye Res. 73 (6): 887-96, 2001), stress, anxiety and prolactin, growth or insulin- And other potential hormonal disorders related to the controlled release of adrenocepticotropin (Francis, K., et al., FASEB J. 77: 2266-2268, 2003; Hansen, TK, Endocrinology 144 (12): 5422-9, 2003).

Two-way communication exists between the immune system and the endocrine system using molecules such as hormones and cytokines. Recently, it has been found that in the new pathway, C3a, complement-inducible cytokines stimulate pituitary hormone release and activate the hypothalamic-pituitary-contractile, response centers for stress responses and response centers that regulate inflammation . C3a receptors are expressed in pituitary-hormone-secreted and non-hormone-secreted (folliculostellate) cells. C3a and C3adesArg (non-inflammatory metabolites) stimulate pituitary cell cultures that release prolactin, growth hormone, and adrenocorticotropin. Serum levels of these hormones, along with adrenocorticosterone, increase the dose in a dose dependent manner in vivo administration of recombinant C3a and C3adesArg. This means that the complement pathway regulates tissue-specific and systemic inflammatory responses through communication with the endocrine pituitary gland (Francis, K., et al., FASEB J. 77: 2266-2268, 2003).

Many studies in animals and humans indicate that growth hormone (GH) and insulin-like growth factor-I (IGF-I) regulate immune function. GH therapy increases the mortality rate of critical patients. Excessive mortality is almost due to septic shock or multi-organ dysfunction, indicating that GH-induced regulation of immune and complement function is involved. Metabolic lectin (MBL) is a plasma protein that plays an important role in congenital immunity through the activation of complement cascade and inflammation after binding of carbohydrate structures. Evidence supports a significant effect on growth hormone-induced MBL levels and thus potentially influences lectin-dependent complement activation (Hansen, T. K., Endocrinology 144 (12): 5422-9, 2003).

Tropaoxidase (TPO) is one of the major autoantigens involved in autoimmune thyroid disease. TPO consists of a large N-terminal myelo-peroxidase-like module according to a complement control protein (CCP) -like module and a epidermal growth factor-like module. The CCP module is a member of molecules involved in the activation of C4 complement components, and studies investigated whether C4 is bound to TPO and activates the complement pathway in autoimmune symptoms. TPO through these CCP modules can directly activate the complement without intervention by Ig. Furthermore, in patients with Hashimoto's thyroiditis, thyroid cells overexert all the lower components of C4 and the complement pathway. These results indicate that TPO may contribute to the mass cell destruction observed in Hashimoto's thyroiditis (Blanchin, S., et al., 2001), along with other mechanisms of activation of the complement pathway.

One aspect of the invention thus provides a method of inhibiting MASP-2-dependent complement activation to treat endocrine disorders by administering to the subject suffering an endocrine disorder a composition comprising a therapeutically effective amount of a MASP-2 inhibitor in a pharmaceutical carrier to provide. Symptoms treated according to the present invention include, but are not limited to, Hashimoto's thyroiditis, stress, anxiety and other potential hormonal disorders related to prolactin, growth or insulin-like growth factor, and regulated release of adrenocorticotropin of the pituitary gland do. MAS-2 inhibitors may be administered to a subject systemically, for example, by oral, buccal, intravenous, intramuscular, inhalation, nasal, subcutaneous or other parenteral administration, or potentially non-peptide. The MASP-2 inhibitor composition may be combined with one or more additional therapeutic agents. Administration can be repeated by the clinician until symptoms improve.

A scientific symptom

Age-related macular degeneration (AMD) is an illness of the eye that affects millions of adults, but biochemical, cellular, and / or molecular sequelae that still develop AMD are not well known. AMD is responsible for the progressive destruction of the macula and involves the formation of cellular deposits called the drusen, located behind and behind the macula and between the retinal pigment epithelium (RPE) and the choroid. Recent studies have found that proteins related to inflammatory and immuno-mediated processes are potent in the DRUGEN-tubule components. Transcripts encoding a number of such molecules are detected in the retina, RPE, and colloidal cells. These data demonstrate that dendritic cells, a potent antigen-presenting cell, are closely related to drusen development and that complement activation is a key pathway in DRUGEN and RPE-choroid interface (Hageman, GS, et. al., Prog. Retin. Eye Res., 20: 705-732, 2001).

Several non-dependent studies have shown a strong association between AMD and genetic polymorphisms in the complement factor H (CFH) gene, and the likelihood of AMD has been increased by a factor of 7.4 within individual homozygotes for allelic disability (Klein , Science 308: 362-364, 2005; Haines et al., Science 308: 362-364. 2005; Edwards et al., Science 308: 263-264, 2005). The CFH gene was mapped to chromosome 1q31, an area of interest in AMD by six independent binding scans (see, for example, D. W. Schultz et al., Hum. Mol. Genet. 72: 3315, 2003). CFH is known as a key regulator of complement systems. It is known that CFH on cells and circulation regulates complement activation by inhibiting the activation of C3 to C3a and C3b and by inactivating the existing C3b. Adherence of C5b-9 is observed in Bruch's membrane, capillary columnar and drusen in AMD patients (Klein et al.). Immunofluorescence experiments indicate that in AMD, the polymorphism of CFH induces complement attachment in colloid capillaries and colloid blood vessels (Klein et al.).

The membrane - associated complement inhibitor, complement receptor 1, was localized in drusen but not immunohistochemically in RPE cells. In contrast, the second membrane-associated complement inhibitor, membrane cofactor protein, is present in the DRG-related RPE cells, and in the small globular structural components in the DRUGEN. This previously unidentified component exhibits a strong immunoreactivity to the proteolytic fragment of the complement component C3 that is characteristically attached to the complement activation site. It is suggested that this structure is presented in the residual debris of RPE cell destruction, which is the target of complement attack (Johnson, L.V., et al., Exp. Eye Res. 73: 887-896, 2001).

The identification and localization of these multiple complement modulators and complement activation products (C3a, C5a, C3b, C5b-9) suggests that chronic complement activation plays an important role in the processes of Druggen biogenesis and AMD pathogenesis (Hageman et al., Progress Retinal Eye Res. 20: 705-32, 2001). Confirmation of the C3 and C5 activation products in Druggen is based on the understanding of the present invention, since both C3 and C5 are common to all three pathways, so that insights into whether the complement is activated through the classical pathway, the lectin pathway or the alternative amplification loop to provide. However, both studies have observed drusen immuno-labeling using antibodies specific for C1q, an essential recognition element for activation of the classical pathway (Mullins et al., FASEB J. 74: 835-846, 2000; Johnson et al., Exp. Eye Res. 70: 441-449, 2000). Both studies concluded that C1q immuno-labeling in drusen was not generally observed. This negative result by C1q means that complement activation in the drusen does not occur through the classical pathway. In addition, immune-labeling of the drug to the immuno-complex components (IgG light chain s, IgM) has been reported to be weak in the study of Mullins et al., 2000, and additionally, Indicating an auxiliary role in activation.

Two recently published studies evaluated the role of complement in the development of laser-induced colloid neovascularization (CNV) in human CNV model mice. Using immunohistochemical methods, Bora and colleagues (2005) found significant attachment of complement activation products C3b and C5b-9 (MAC) in neovascular complexes after laser treatment (Bora et al., J. Immunol. 174: 491-1, 2005). Importantly, CNV is not developed in C3 deficient mice (C3 - / - mice), an essential component of all complement activation pathways. The RNA messenger levels of VEGF, TGF-β2, and β-FGF, the three angiogenic factors involved in CNV, were increased in mouse eye tissue after laser-induced CNV. Significantly, complement depletion resulted in a record reduction in the RNA level of these angiogenic factors.

Using ELISA, Nozaki and co-workers have shown that potential anapylatoxins C3a and C5a occurred early in the course of laser-induced CNV (Nozaki et al., Proc. Natl. Acad. : 2328-33, 2006). In addition, these two bioactive fragments of C3 and C5 induced VEGF expression after intravitreal injection in wild type mice. Consistent with these results, Nozaki and co-workers have also found that genetic removal of C3a and C5a receptors reduces VEGF expression and CNV formation after laser injury, and antibody-mediated neutralization of C3a or C5a or pharmacological blockade of these receptors . Previous studies have established that the use of leukocytes and macrophages in particular plays a central role in laser-induced CNV (Sakurai et al., Invest. Opthomol. Vis. ScL 44: 3578-85, 2003; Espinosa-Heidmann, et al., Invest. Opthomol. Vis., ScL 44: 3586-92, 2003). In their 2006 paper, Nozaki and colleagues reported that leukocyte use was record- edly reduced in C3aR (- / -) and C5aR (- / -) mice after laser injury.

One aspect of the present invention is therefore to provide a method of treating a subject suffering from age-related macular degeneration or other complement mediated disease, such as a macular degeneration or other complement mediated disease, by administering to the subject a composition comprising a therapeutically effective amount of a MASP- Provides a method of inhibiting MASP-2-dependent complement activation to treat scientific symptoms. The MASP-2 inhibitory composition can be administered locally to the eye by, for example, perfusion or application of a composition in the form of a gel, ointment or drop. Alternatively, the MASP-2 inhibitor can be administered to a subject systemically, for example, by oral, buccal, intravenous, intramuscular, inhalation, nasal, subcutaneous or other parenteral administration, or potentially non-peptide. MASP-2 inhibitor compositions can be combined with one or more additional therapeutic agents, such as those described in U.S. Pat. Patent Application Publication No. 2004-0072809-A1. Administration may be repeated by the clinician until the symptoms improve or are controlled.

IV. MASP-2 inhibitor

As one aspect of the present invention, there is provided a method of inhibiting the adverse effect of the MASP-2-dependent complement activation of the present invention. The MASP-2 inhibitor is administered in an amount effective to inhibit MASP-2-dependent complement activation in a living subject. Representative MASP-2 inhibitors include: molecules that inhibit the biological activity of MASP-2 (such as a small molecule inhibitor, an anti-MAST-2 antibody or MASP-2 or protein- (E.g., MASP-2 antisense nucleic acid molecules, MASP-2 specific RNAi molecules, and MASP-2 ribozymes) that decrease the expression of MASP-2, Prevent activation of the complement pathway. MASP-2 inhibitors may be used alone or in combination with other therapeutic agents as adjunctive therapies to enhance the therapeutic benefits of other medical treatments.

The inhibition of MASP-2-dependent complement activation is characterized by at least one of the following changes in the components of the complement system that occur as a result of administration of a MASP-2 inhibitor according to the methods of the present invention: MASP-2- Inhibition of the production or production of products C4b, C3a, C5a and / or C5b-9 (MAC) (as measured, for example, as described in Example 2), hemolysis measurements using non- sensitized levitt or guinea pig red- (For example, as described in Example 2), or a decrease in C3 cleavage and C3b attachment (e.g., as described in Example 2), reduction of C4 cleavage and C4b attachment Lt; / RTI &gt;

In accordance with the present invention, MASP-2 inhibitors effective to inhibit the MASP-2-dependent complement activation system are used. MASP-2 inhibitors used in the features of the invention include, for example, anti-MAST-2 antibodies and fragments thereof, MASP-2 inhibitory peptides, small molecules, MASP-2 soluble receptors and inhibitors of expression. MASP-2 inhibitors may inhibit the MASP-2-dependent complement activation system by blocking the biological function of MASP-2. For example, inhibitors can effectively block MASP-2 protein-to-protein interactions, block MASP-2 dimerization or binding, block block Ca2 + binding, interfere with MASP-2 serine protease activation sites, Or MASP-2 protein expression.

In one embodiment, the MASP-2 inhibitor selectively inhibits only MASP-2 complement activation without compromising the function of the C1q-dependent complement activation system.

In one embodiment of the invention, the MASP-2 inhibitor useful in the methods of the present invention is a specific inhibitor that specifically binds to a polypeptide comprising SEQ ID NO: 6 with an affinity that is at least ten times greater than other antigens in the complement system Gt; MASP-2 &lt; / RTI &gt; inhibitor. In another embodiment, the MASP-2 inhibitor specifically binds to a polypeptide comprising SEQ ID NO: 6 with at least 100-fold greater binding affinity than other antigens in the complement system. The binding affinity of a MASP-2 inhibitor may be determined using suitable binding assays.

MASP-2 polypeptides exhibit similar molecular structures to proteases of MASP-1, MASP-3, and C1r and C1s, C1 complement systems. The cDNA molecule in SEQ ID NO: 4 encodes a representative example of MASP-2 (consisting of the amino acid sequence in SEQ ID NO: 5), a leader sequence (aa 1-15) that is cleaved after secretion into the human MASP- To provide the mature form of human MASP-2 (SEQ ID NO: 6). As shown in Figure 2, the human MASP2 gene contains 12 exons. The human MASP-2 cDNA is encoded by exons B, C, D, F, G, H, I, J, The alternative splice is a 2O kDa protein ("MApl9 ", or" sMAP, referred to as MBL-linked protein 19) encoded by the generation of exons B, C, D and E (SEQ ID NO: ") (SEQ ID NO: 2). The cDNA molecule of SEQ ID NO: 50 encodes murine MASP-2 (consisting of the amino acid sequence of SEQ ID NO: 51) and provides a murine MASP-2 polypeptide secreted, To become the mature form (SEQ ID NO: 52). The cDNA molecule of SEQ ID NO: 53 encodes rat MASP-2 (consisting of the amino acid sequence in SEQ ID NO: 54) and provides a leader sequence that is cleaved after secretion into the rat MASP-2 polypeptide, To become the mature form (SEQ ID NO: 55).

Those skilled in the art will be able to identify the single alleles of human, murine and retest MASP-2, and the sequences disclosed in SEQ ID NO: 4, SEQ ID NO: 50 and SEQ ID NO: 53 which represent opposite variants and alternative splicing It is understood that sequences may occur. Allelic variants of the nucleotide sequence shown in SEQ ID NO: 4, SEQ ID NO: 50 and SEQ ID NO: 53, including those involving silent mutations and mutations causing amino acid sequence changes, are within the scope of the present invention. Allelic variants of the MASP-2 sequence can be cloned by probing the cDNA or genomic library of another individual according to standard procedures.

The domain of the human MASP-2 protein (SEQ ID NO: 6) is shown in Figure 3A and comprises the N-terminal Clr / C1s / sea urchin Vegf / bone forming protein (CUBI) domain (aa 1-121, SEQ ID NO: 6 ), The epidermal growth factor-like domain (aa 122-166), the second CUBI domain (aa 167-293), and the tandem and serine protease domains of the complement control protein domain. Substitution splicing of the MASP 2 gene becomes MAp 19 (Figure 3B). MAp 19 is a non-enzymatic protein containing the N-terminal CUBI-EGF region of MASP-2 with four additional residues (EQSL) derived from exon E as shown in Fig.

Several proteins appear to bind or interact with MASP-2 through protein-to-protein interactions. For example, MASP-2 is known to bind to or form Ca2 + -dependent complexes, the lectin proteins MBL, H-picoline and L-picoline. Each MASP-2 / lectin complex is known to activate complement through MASP-2-dependent cleavage of proteins C4 and C2 (Ikeda, K., et al., J. Biol. Chem. 262: 7451-7454, Matsushita, M., et al, J. Exp. Med., 776: 1497-2284, 2000; Matsushita, M., et al., J. Immunol., 768: 3502-3506, 2002). Studies have shown that the CUBI-EGF domain of MASP-2 is essential for the binding of MASP-2 and MBL (Thielens, N. M., et al., J. Immunol. 766: 5068, 2001). This indicates that the CUBIEGFCUBII domain mediates dimerization of MASP-2, which requires the formation of an active MBL complex (Wallis, R., et al., J. Biol. Chem. 275: 30962-30969, . Thus, MASP-2 inhibitors may be found to bind to or interfere with the MASP-2 target region known to be important for MASP-2-dependent complement activation.

Anti-MASP-2 antibody

In another aspect of the invention, the MASP-2 inhibitor comprises an anti-MASP-2 antibody that inhibits the MASP-2-dependent complement activation system. Anti-MASP-2 antibodies useful in characterizing the present invention include polyclonal, monoclonal or recombinant antibodies derived from all antibody-producing mammals and include, but are not limited to, multispecific, chimeric, human adaptive, anti- Lt; / RTI &gt; Antibody fragments include Fab, Fab ', F (ab) 2, F (ab') 2, Fv fragments, scFv fragments and further single-chain antibodies described herein.

Several anti-MASP-2 antibodies have been described herein, some of which are listed in Table 1 below. These previously described anti-MASP-2 antibodies were screened for their ability to inhibit the MASP-2-dependent complement activation system using the assays described herein. For example, the anti-retroviral MASP-2 Fab2 antibody is identified by blocking MASP-2 dependent complement activation, which is described in more detail in Examples 24 and 25. Once the function of the anti-MASP-2 antibody as a MASP-2 inhibitor is identified, it can be used for the production of an anti-idiotypic antibody described below and for the identification of other MASP-2 binding molecules.

Table 1: MASP-2 specific antibodies herein antigen Antibody type Resources Recombinant MASP-2 Rettockon Peterson, S. V., et al., Mol. Immunol. 37: 803-811,2000 Recombinant human CCP1 / 2-SP fragment (MoAb 8B5) Leto MoAb (subclass IgG1) Moller-Kristensen, M., et al., J. of Immunol. Methods 282: 159-167, 2003 Recombinant human MAp19 (MoAb 6G12) (cross reacted with MASP-2) Leto MoAb (subclass IgG1) Moller-Kristensen, M., et al., J. of Immunol. Methods 282: 159-167, 2003 hMASP-2 Mouse MoAb (S / P) mouse MoAb (N-terminal) Peterson, S. V., et al., Mol. Immunol. 35: 409, April 1998 hMASP-2 (CCP1-CCP2-SP domain) Ret MoAb: produced in Nimoab 101, hybridoma cell line 03050904 (ECACC) WO 2004/106384 hMASP-2 (full-length his tag) (DSMZ) Nimoab101 hybridoma cell line M0545YM029 (DSMZ) Nimoab 109 hybridoma cell line M0545YM046 (DSMZ) Nimoab 110 hybridoma cell line M0545YM035 (DSMZ) WO 2004/106384

Anti-MASP-2 antibody with reduced effector function

In another aspect of the invention, the anti-MASP-2 antibody possesses reduced effector function to reduce inflammation that can lead to activation of the classical complement pathway. The ability of the IgG molecule to trigger the classical complement pathway is known to be present in residues within the Fc portion of the molecule (Duncan, A. R., et al., Nature 332: 738-740 1988). IgG molecules in which the Fc portion of the molecule is cleaved by enzymatic cleavage lack this effector function (Harlow, Antibodies: A Laboratory Manual, Cold Spring Harbor Laboratory, New York, 1988). Thus, antibodies with reduced effector function were generated as a result of deficiency of the Fc portion of the molecule by minimizing effector function, or by retaining a genetically engineered Fc sequence that is a human IgG2 or IgG4 isotype.

Antibodies with reduced effector function are described in Example 9 of the present application and in Jolliffe, et al., Int'l Rev. Immunol. 70: 241-250, 11993, and Rodrigues, et al., J. Immunol. &Lt; / RTI &gt; 151: 6954-6961, 1998. Antibodies with reduced effector function include human IgG2 and IgG4 isotypes that reduce the ability to activate complement and / or interact with Fc receptors (Ravetch, JV, et al., Annu. Rev. Immunol. 9: 457-492, 1991; Isaacs, JD, et al., J. Immunol. 148: 3062-3011, 1992; van de Winkel, JG, et al., Immunol., 74: 215-221, 1993). Human adaptive or whole human antibodies specific for human MASP-2, including IgG2 or IgG4 isotypes, can be produced by one of a number of methods known to those of skill in the art, see Vaughan, TJ, et al. , Nature Biotechnical 16: 535-539, 1998.

Production of anti-MASP-2 antibody

Anti-MAST-2 antibodies can be produced using MASP-2 polypeptides (e.g., full length MASP-2) or antigenic MASP-2 epitope-containing peptides (e.g., portions of MASP-2 polypeptides). The immunogenic peptides are as small as 5 amino acid residues. For example, a MASP-2 polypeptide comprising the entire amino acid sequence of SEQ ID NO: 6 may be used to derive an anti-MAST-2 antibody useful in the methods of the invention. Specific MASP-2 domains known to be involved in protein-protein interactions, such as CUBI and CUBIEGF domains, and regions containing serine-protease activation sites, are expressed as recombinant polypeptides used as antigens as described in Example 5 do. In addition, peptides containing at least six amino acid portions of the MASP-2 polypeptide (SEQ ID NO: 6) are useful for inducing MASP-2 antibodies. Additional examples of MASP-2 inducing antigens inducing MASP-2 antibodies are provided in Table 2 below. MASP-2 peptides and polypeptides used to generate antibodies are isolated as native polypeptides, or as recombinant or synthetic peptides and as catalytically inactive recombinant polypeptides, such as MASP-2A. This is further explained in Examples 5-7. In another aspect of the invention, anti-MAST-2 antibodies were obtained using the gene insertion mouse strains described in Examples 8 and 9 and described below.

Antigens useful for producing anti-MAST-2 antibodies may include fusion polypeptides, such as fusion of MASP-2 or immunoglobulin polypeptides or portions thereof having a maltose-binding protein. Polypeptide immunogens can be full length molecules or parts thereof. If the polypeptide moiety is hapten-like, then this moiety is preferably linked or linked to the macromolecular carrier (e.g., a heat-labled clam hemostatic (KLH), bovine serum albumin (BSA) or tetanus toxoid) for immunization .

Table 2: MASP-2 induced antigen SIQ ID NO: Amino acid sequence SIQ ID NO: 6 Human MASP-2 protein SIQ ID NO: 51 Murine MASP-2 protein SIQ ID NO: 8 The CUBI domain of human MASP-2 (SIQ ID NO: 6 of aa 1-121) SIQ ID NO: 9 The CUBIEGF domain of human MASP-2 (SIQ ID NO: 6 of aa 1-166) SIQ ID NO: 10 The CUBIEGFCUBII domain of human MASP-2 (SIQ ID NO: 6 of aa 1-293) SIQ ID NO: 11 The EGF domain of human MASP-2 (SIQ ID NO: 6 of aa 122-166) SIQ ID NO: 12 The serine-protease domain of human MASP-2 (SIQ ID NO: 6 of aa 429-671) SIQ ID NO: 13 GKDSCRGDAGGALVFL Serine-protease inactivated mutants (SIQ ID NO: 6 of aa 610-625, mutant Ser 618) SIQ ID NO: 14 TPLGPKWPEPVFGRL Human CUBI peptide SIQ ID NO: 15 TAPPGYRLRLYFTHFDLEL SHLCEYDFVKLSSGAKVL ATLCGQ Human CUBI peptide SIQ ID NO: 16 TFRSDYSN The MBL binding region in the human CUBI domain SIQ ID NO: 17 FYSLGSSLDITFRSDYSNEK PFTGF The MBL binding region in the human CUBI domain SIQ ID NO: 18 IDECQVAPG EGF peptide SIQ ID NO: 19 ANMLCAGLESGGKDSCRG DSGGALV Peptides of serine-protease active sites

Polyclonal antibody

Polyclonal antibodies to MASP-2 can be prepared by immunizing an animal with an MASP-2 polypeptide or an immunogenic portion thereof using methods known to those skilled in the art. See, e. G., Green, et al., "Production of Polyclonal Antisera" in Immunochemical Protocols (Manson, ed.), Page 105 and further described in Example 6. The immunogenicity of the MASP-2 polypeptides can be determined by the use of mineral gels such as aluminum hydroxide or Freund's adjuvant (complete or incomplete), surface active substances such as lyso lecithin, pluronic polyols, next ions, oil emulsions, Lt; RTI ID = 0.0 &gt; and / or &lt; / RTI &gt; dinitrophenol. Polyclonal antibodies are generally generated in animals such as horses, cows, dogs, chickens, rats, mice, levitra, guinea pigs, chlorine, or sheep. Alternatively, anti-MASP-2 antibodies useful in the present invention can be derived from sub-human primates. General techniques for the generation of diagnostically and therapeutically useful antibodies in baboons are described, for example, in Goldenberg et al., International Patent Publication No. &lt; RTI ID = 0.0 &gt; WO 91/11465, and in Losman, M. J., et al., Int. J. Cancer 46: 310, 1990. Serum containing immunologically active antibodies is produced from the blood of such immunized animals using known standard procedures.

Monoclonal antibody

In one embodiment, the MASP-2 inhibitor is an anti-MASP-2 monoclonal antibody. Anti-MASP-2 monoclonal antibodies are highly specific for a single MASP-2 epitope. As used herein, a variant "monoclonal" is obtained from a substantially homogeneous population of antibodies and exhibits characteristics of the antibody that can not be accounted for as an antibody required production by any particular method. Monoclonal antibodies can be obtained using any technique that provides for the production of antibody molecules by continuous cell lines in culture, such as hybridization magic in Kohler, G., et al., Nature 256: 495, 1975 , They may be produced by a recombinant DNA method (see, for example, US Patent No. 4,816,567, Cabilly). Monoclonal antibodies can be isolated from phage antibody libraries, which are described in Clackson, T., et al., Nature 352: 624-628, 1991, and Marks, J. D., et al., J. Mol. Biol. 222: 581-597, 1991. Such antibodies can be any immunoglobulin class, including IgG, IgM, IgE, IgA, IgD, and all subclasses thereof.

For example, a monoclonal antibody can be obtained by injecting a composition comprising a MASP-2 polypeptide or portion thereof into a suitable mammal (e.g., BALB / c mouse). After a predetermined time, spleen cells were removed from the mice and suspended in cell culture medium. Splenocytes were fused to infinitely proliferating cell lines to form hybridomas. The hybridomas formed were selected for their ability to grow in cell culture and produce monoclonal antibodies to MASP-2. Additional examples illustrating the production of anti-MAST-2 mAb are provided in Example 7 (Current Protocols in Immunology, Vol. L, John Wiley & Sons, pages 2.5.1-2.6.7, 1991.).

Human monoclonal antibodies can be obtained using transgenic mice engineered to produce specific human antibodies in response to antigen testing. In this technique, the elements of the human immunoglobulin heavy chain and the light chain locus were introduced into mouse strains derived from embryonic stem cell lines containing the targeted cleavage of the endogenous immunoglobulin heavy chain and light chain locus. Gene-injected mice can, for example, synthesize human antibodies specific for human antigens that are MASP-2 antigens, and mice use the conventional Kollar-Milstron technique described further in Example 7 to generate B - &lt; / RTI &gt; cells to produce human MASP-2 antibody-secreted hybridomas. Gene insertion mice of the human immunoglobulin genome are available (e.g., from Abgenix, Inc., Fremont, CA, and Medarex, Inc., Annandale, N. J.). Methods for obtaining human antibodies in gene-injected mice are described, for example, in Green, L. L., et al., Nature Genet. 7:13, 1994; Lonberg, N., et al., Nature 368: 856, 1994; And Taylor, L. D., et al., Int. Immun. 6: 579, 1994.

Monoclonal antibodies can be isolated and purified from hybridoma cultures by a variety of established techniques. Such separation techniques include affinity chromatography with protein-A sepharose, size-exclusion chromatography, and ion-exchange chromatography (see, for example, Coligan at pages 2.7.1-2.7.12 and pages 2.9 Humana Press, Inc., Vol. 10, pages 79-104, 1992). &Quot; Purification of Immunoglobulin G (IgG) ", in Methods in Molecular Biology, Humana Press, Inc., Vol. If produced, polyclonal, monoclonal or phage-inducible antibodies are tested for specific MASP-2 binding. Various assays known to those skilled in the art are used to detect antibodies specifically bound to MASP-2. Exemplary assays include immunoprecipitation assays (e. G., Ausubel et al.) By Western blot or standard methods, immunoelectrophoresis, enzyme-linked immunosorbent assay, dot blot, inhibition or competitive assay and sandwich assay (Harlow and Land , Antibodies: A Laboratory Manual, Cold Spring Harbor Laboratory Press, 1988). When the antibody is known to specifically bind to MASP-2, the anti-MAASP-2 antibody can be assayed by several assays, such as lectin-specific C4 cleavage assays (Example 2), C3b attachment assays ) Or the ability to function as a MASP-2 inhibitor in the C4b attachment assay (Example 2).

The affinity of anti-MAST-2 mAb can be readily determined by those skilled in the art (see, for example, Scatchard, A., NY Acad. ScL 57: 660-672, 1949). In one embodiment of the invention, the anti-MAST-2 monoclonal antibody useful in the present invention binds to MASP-2 with a binding affinity of < 100nM, preferably <10nM and most preferably <2nM.

Chimeric / human adaptive antibody

Monoclonal antibodies useful in the methods of the present invention are those in which the portion of the heavy chain and / or light chain matches a corresponding sequence in an antibody derived from a particular species or an antibody belonging to a particular antibody class or subclass, or the remainder of the homologue or chain (s) Antibodies derived from another species or another antibody class or subclass antibody, and chimeric antibodies that are identical or homologous to corresponding sequences within fragments of such antibodies (US Patent No. 4,816,567 to Cabilly, and Morrison, SL, et al., Proc Natlagad, ScL USA 87: 6851-6855, 1984).

One form of chimeric antibody useful in the present invention is the human adaptive monoclonal anti-MAST-2 antibody. Human adaptive non-human (e.g., murine) antibodies are chimeric antibodies, which contain minimal sequences derived from non-human immunoglobulins. Human Adaptive Monoclonal Antibodies are produced by transferring non-human (e.g., murine) complementarity determining regions (CDRs) from the heavy and light chain variable chains of mouse immunoglobulins to human variable domains. In general, residues of human antibodies have been substituted within the framework regions of non-human counterparts. In addition, the human adaptive antibody may comprise an acceptor antibody or a residue found in the donor antibody. Such modifications are made to purify antibody performance. In general, a human adaptive antibody will comprise substantially all at least one, and generally two variable domains, wherein all or substantially all of the hypervariable loops correspond to those of a non-human immunoglobulin and all or substantially all of the Fv The framework region is of the human immunoglobulin sequence. The human adaptive antibody optionally comprises at least a portion of that of an immunoglobulin constant region (Fc), typically a human immunoglobulin. For more information, see Jones, P. T., et al., Nature 321: 522-525, 1986; Reichmann, L., et al., Nature 332: 323-329, 1988; And Presta, Curr. Op. Struct. Biol. 2: 593-596, 1992.

Human adaptive antibodies useful in the present invention include human monoclonal antibodies comprising at least a MASP-2 binding CDR3 region. Together, the Fc portion was replaced to produce IgA or IgM and human IgG antibodies. These human adaptive antibodies have specific clinical uses because they specifically recognize human MASP-2 but do not cause an immune response against the antibody itself in humans. As a result, studies involving human in vivo administration should be made, especially when repeated or long-term administration is required.

An example of the production of a human adaptive anti-MASP-2 antibody in a murine anti-MASP-2 monoclonal antibody is provided in Example 10 herein. Human adaptive monoclonal antibody production techniques are described, for example, in Jones, P. T., et al., Nature 321: 522, 1986; Carter, P., et al., Proc. Natl Acad. ScL USA 89: 4285, 1992; Sandhu, J. S., Crit. Rev. Biotech. 72: 437,1992; Singer, LL, et al., J. Immun. 750: 2844,1993; Sudhir (ed.), Antibody Engineering Protocols, Humana Press, Inc., 1995; Kelley, "Engineering Therapeutic Antibodies, " in Protein Engineering: Principles and Practice, Cleland et al. (eds.), John Wiley & Sons, Inc., pages 399-434, 1996; U.S.A. Patent No. 5,693, 762, Queen, 1997. In addition, there are manufacturers that synthesize human adaptive antibodies in the size-specific murine antibody region, such as Protein Design Lab (Mountain View, CA).

Recombinant antibody

Anti-MAST-2 antibodies may also be prepared using recombinant methods. For example, a human antibody can be conjugated to a human immunoglobulin expression library (e.g., Stratagene, Corp., La Jolla, CA) to produce a fragment of a human antibody (VH, VL, FV, Fd, Fab or F , Obtained from CA). These fragments can be used to construct whole human antibodies using techniques similar to those for producing chimeric antibodies.

Anti-idiotype antibody

If the anti-MAST-2 antibody is found to have the desired inhibitory activity, such an antibody may be used to generate an anti-idiotype antibody similar to that of MASP-2 using techniques known in the art . See, for example, Greenspan, N. S., et al., FASEB J. 7: 437, 1993. For example, antibodies that competitively inhibit the MASP-2 protein interaction that binds to MASP-2 and are required for complement activation may resemble the MBL binding site on the MASP-2 protein and may bind to MASP-2, Can be used to bind to the binding ligand and generate the neutralizing anti-idiotate.

Immunoglobulin fragment

MASP-2 inhibitors useful in the methods of the present invention include but are not limited to intact immunoglobulin molecules as well as Fab, Fab ', F (ab) 2, F (ab') 2 and Fv fragments, scFv fragments, dimers, Antibody molecule and a multispecific antibody formed on the antibody fragment.

It is known in the art that only a small portion of the antibody molecule, the paratope, is involved in the binding of antibodies and their epitopes (see, for example, Clark, WR, The Experimental Foundations of Modern Immunology, Wiley & NY, 1986). The pFc 'and Fc regions of the antibody are the effector of the classical complement pathway, but do not participate in antigen binding. An antibody in which the pFc 'region is enzymatically cleaved or produced without the pFc' region is designated as the F (ab ') 2 fragment and retains all of the antigen binding sites of the intact antibody. Isolated F (ab ') 2 fragments are referred to as bivalent monoclonal fragments by their two antigen binding sites. Similarly, antibodies in which the Fc region is enzymatically cleaved or produced without the Fc region are designated Fab fragments and retain one of the antigen binding sites of the intact antibody molecule.

Antibody fragments can be obtained by proteolytic hydrolysis of whole antibodies by pepsin or papain digestion by conventional methods. For example, the antibody fragment is produced by enzymatic cleavage of the antibody with pepsin to provide a 5S fragment denoted F (ab ') 2. This fragment can be further cleaved using a thiol reducing agent to produce a 3.5S Fab '1 fragment. Optionally, the cleavage reaction can be carried out using a circuit breaker for the sulfhydryl group which is the result of cleavage of the disulfide bond. Alternatively, enzymatic cleavage with pepsin directly produces two univalent Fab fragments and Fc fragments. Such a method is described, for example, in U.S. Pat. Patent No. 4,331,647 to Goldenberg; Nisonoff, A., et al., Arch. Biochem. Biophys. 89: 230, 1960; Porter, R. R., Biochem. J. 73: 119, 1959; Edelman, et al., In Methods in Enzymology, 1: 422, Academic Press, 1967; And by Coligan at pages 2.8.1-2.8.10 and 2.10.-2.10.4.

In one embodiment, the use of an antibody fragment in which the Fc region is absent is desirable to avoid activation of the classical complement pathway initiated by binding of the Fc and Fc [gamma] receptors. There are several methods that can produce MoAbs that avoid Fc [gamma] receptor interactions. For example, the Fc region of a monoclonal antibody can be chemically removed (e. G., Antisense) by digestion of the proteolytic enzyme and thus, for example, an antigen-binding antibody fragment, such as an Fab or F (Mariani, M., et al., Mol. Immunol. 28: 69-11,1991). Alternatively, a human gamma 4 IgG isotype that does not bind to an Fc [gamma] receptor can be used in the construction of the human adaptive antibodies described herein. Antibodies, single chain antibodies and antigen-binding domains lacking the Fc domain may be engineered using recombinant techniques of the present invention.

Single-chain antibody fragment

Alternatively, a single peptide chain-binding molecule specific for MASP-2, to which heavy and light chain Fv regions are linked, can be generated. Fv fragments can be joined by a peptide linker to form a single-chain antigen binding protein (scFv). Such a single-chain antigen binding protein can be prepared by constructing a structural gene comprising a DNA sequence encoding the VH and VL domains linked by oligonucleotides. The structural gene is inserted into an expression vector, which is subsequently inserted into a host cell, e. Is introduced into E. coli. Recombinant host cells synthesize a single polypeptide chain with a linker peptide linking the two V domains. Methods for producing scFvs are described, for example, in Whitlow, et al., "Method: A Companion to Methods in Enzymology" 2:97, 1991; Bird, et al., Science 242: 423, 1988; U.S.A. Patent No. 4,946,778, Ladner; Pack, P., et al., Bio / Technology 77: 1271, 1993.

As an illustrative example, a MASP-2 specific scFv can be obtained by exposing an in vitro lymphocyte to a MASP-2 polypeptide and selecting an antibody display library within the phage or pseudo-vector (e.g., Using a labeled MASP-2 protein or peptide). The gene encoding the polypeptide having the potential MASP-2 polypeptide binding domain may be a phage or a bacterium, e. Can be obtained by screening randomized peptide libraries displayed on E. coli. Such a random peptide display library can be used to screen for peptides that interact with MASP-2. Techniques for generating and screening randomized peptide display libraries are known in the art (US Patent No. 5,223,409, Lardner; US Patent No. 4,946,778, Ladner; US Patent No. 5,403,484, Lardner; US Patent No. 5,571,698, Lardner And Kay et al., Phage Display of Peptide and Protein Academic Press, Inc., 1996) and random peptide display libraries and such library screening kits can be obtained, for example, from CLONTECH Laboratories, Inc. (Palo Alto, Calif. ), Invitrogen Inc. (San Diego, Calif.), New England Biolabs, Inc. (Beverly, Mass.), And Pharmacia LKB Biotechnology Inc. (Piscataway, NJ).

Another form of anti-MASP-2 antibody fragment useful in characterizing the invention is a peptide that binds to an epitope on the MASP-2 antigen and encodes a single complementary-determining region (CDR) that inhibits MASP-2- to be. A CDR peptide ("minimal recognition unit") can be obtained by constructing a gene encoding the CDR of an antibody of interest. Such a gene can be produced, for example, using a polymerase chain reaction to synthesize an RNA-variable region of an antibody-producing cell (see, for example, Larrick et al., Method: A Companion to Methods in Enzymology Genetic Manipulation of Monoclonal Antibodies in Monoclonal Antibodies: Production, Engineering and Clinical Application, Ritter et al. (Eds.), Page 166, Cambridge University Press, 1995; and Ward et &lt; RTI ID = 0.0 &gt; al., " Genetic Manipulation and Expression of Antibodies, " in Monoclonal Antibodies: Principles and Applications, Birch et al. (eds.), page 137, Wiley-Liss, Inc., 1995).

The MASP-2 antibodies herein are administered to a subject in need thereof to inhibit MASP-2-dependent complement activation. In one embodiment, the MASP-2 inhibitor is a high-affinity human or human adapted monoclonal anti-MAST-2 antibody with reduced effector function.

Peptide inhibitor

In another aspect of the invention, the MASP-2 inhibitor comprises a separate MASP-2 peptide inhibitor comprising an isolated natural peptide inhibitor and a synthetic peptide inhibitor that inhibits the MASP-2-dependent complement activation system. As used herein, the term "isolated MASP-2 peptide inhibitor" refers to a molecule that binds to another recognition molecule (e.g., MBL, H-picoline, M-picoline, or L-picoline) Dependent dependent complement by binding directly to MASP-2 to inhibit MASP-2-dependent complement activation that competes with MASP-2 for MASP-2 and / or is substantially pure and essentially free of other substances Refers to a peptide that inhibits activation.

Peptide inhibitors can be successfully used in vivo to interfere with protein-protein interactions and catalytic sites. For example, peptide inhibitors that adhere to structurally related molecules in LFA-I have recently been approved for clinical use of coagulopathy (Ohman, E.M., et al., European Heart J. 16: 50-55, 1995). Short linear peptides (<30 amino acids) are described as preventing or interfering with integrin-dependent attachment (Murayama, O., et al., J. Biochem. 720: 445-51, 1996). Long peptides ranging in length from 25 to 200 amino acid residues have been successfully used to block integrin-dependent attachment (Zhang, L., et al., J. Biol. Chem. 27i (47): 29953-57, 1996). Generally, long peptide inhibitors have a higher affinity and / or a lower off-rate than short peptides and are therefore more potent inhibitors. Cyclic peptide inhibitors appear to be effective inhibitors of integrin in vivo for the treatment of human inflammatory diseases (Jackson, D.Y., et al., J. Med. Chem. 40: 3359-68, 1997). One of the methods of producing cyclic peptides involves the synthesis of peptides, wherein the terminal amino acid of the peptide is cysteine, so that the peptide is present in the cyclic form by a disulfide bond between the terminal amino acids. Which appears to enhance in vivo affinity and half-life for the treatment of hematopoietic neoplasms (e.g., U.S. Patent No. 6,649,592, Larson).

Synthetic MASP-2 peptide inhibitor

MASP-2 inhibitory peptides useful for use in the methods of the invention are exemplified by amino acid sequences that mimic target regions that are important for MASP-2 function. The inhibitory peptides used in the present invention range in size from about 5 amino acids to about 300 amino acids. Table 3 provides a list of exemplary inhibitory peptides used in the methods of the invention. The candidate MASP-2 inhibitory peptides were tested for their ability to function as MASP-2 inhibitors with one of several assays, such as lectin-specific C4 cleavage assay (Example 2) and C3b attachment assay (Example 2) .

In one embodiment, the MASP-2 inhibitory peptide is derived from a MASP-2 polypeptide and is selected from the full-length maturation MASP-2 protein (SEQ ID NO: 6), or a particular domain of a MASP-2 protein, (SEQ ID NO: 8), the CUBIEGF domain (SEQ ID NO: 9), the EGF domain (SEQ ID NO: 11), and the serine protease domain (SEQ ID NO: 12). As previously described, the CUBEGFCUBII region appears to be required for dimerization and association with MBL (Thielens et al., Supra). In particular, the peptide sequence TFRSDYN (SEQ ID NO: 16) in the CUBI domain of MASP-2 involved MBL binding in studies identifying humans with homozygous mutations from Asp105 to Gly105, leading to loss of MASP-2 in the MBL complex (Stengaard-Pedersen, K., et al., New England J. Med. 349: 554-560, 2003).

The MASP-2 inhibitory peptide may be derived from MAp19 (SEQ ID NO: 3). As described in Example 30, MAp19 (SEQ ID NO: 3) (sMAP) possesses the ability to down-regulate the lectin pathway, which is activated by the MBL complex. Iwaki et al., J. Immunol. 777: 8626-8632, 2006. While it is expected to be bound by theory, sMAP appears to be able to occupy the MASP-2 / sMAP binding site within MBL and prevents MASP-2 from binding to MBL . It has been reported that sMAP competes with picoline to compete with MASP-2 and inhibits complement activation by picoline A / MASP-2 complex. Endo Y. et al., Immunogenetics 57: 837-844 (2005).

In one embodiment, the MASP-2 inhibitory peptide is derived from a lectin protein that binds to MASP-2 and is involved in the lectin complement pathway. It has been found that several different lectins are involved in this pathway, including mannan-binding lectin (MBL), L-picoline, M-picoline and H-picoline. Matsushita, M., et al., J. Exp. Med., 776: 1497-2284, 2000; Matsushita, M., et al., J. Biol. Chem. 262: 7451-7454 , et al., J. Immunol. 168: 3502-3506, 2002). These lectins are present in the serum as oligomers of homotrimeric subunits, each carrying an N-terminal collagen-like fiber with a carbohydrate recognition domain. These other lectins have been shown to bind to MASP-2, and the lectin / MASP-2 complex activates complement through cleavage of proteins C4 and C2. P-choline has an amino-terminal region of 24 amino acids, a collagen-like domain of 11 Gly-Xaa-Yaa repeats, a neck domain of 12 amino acids, and a fibrinogen-like domain of 207 amino acids (Matsushita, M., et al., J. Immunol. 168: 3502-3506, 2002). H-picoline binds to GIcNAc; S. typhimurium, S. &lt; RTI ID = 0.0 &gt; Minnesota (S. Coagulate human red blood cells coated with LPS derived from E. coli. H-picoline has been shown to bind to MASP-2 and MAp 19 and to activate the lectin pathway. Id. L-picoline / P35 also binds to GIcNAc and binds to MASP-2 and MAp 19 in human serum, and this complex has been shown to activate the lectin pathway (Matsushita, M., et al., J Immunol., 164: 2281,2000). Thus, the MASP-2 inhibitory peptides used in the present invention may be selected from the group consisting of MBL protein (SEQ ID NO: 21), H-picoline protein (Genbank accession number NM_173452), M- picoline protein (Genbank accession number 000602) It contains a region of at least five amino acids selected from the choline protein (Genbank accession number NM_015838).

More specifically, the scientists found that the MASP-2 binding site on MBL contained 12 GIy-XY triplets "GKD GRD GTK GEK GEP GQG LRG LQG POG KLG &lt; / RTI &gt; between the hinge and neck of the C-terminal portion of the collagen- (SEQ ID NO: 26) (Wallis, R., et al., J. Biol. Chem. 279: 14065, 2004). This MASP-2 binding site region was highly converted in human H-picoline and human L-picoline. The consensus binding site is present in all three lectin proteins including the amino acid sequence "OGK-X-GP" (SEQ ID NO: 22), where the letter "O" refers to hydroxyproline and the letter "X" (Wallis et al., 2004, supra). Thus, in one embodiment, the MASP-2 inhibitory peptide useful in the present invention is at least 6 amino acids in length and comprises SEQ ID NO: 22. MBL-derived peptides containing the amino acid sequence "GLR GLQ GPO GKL GPO G" (SEQ ID NO: 24) were found to bind MASP-2 in vitro (Wallis, et al., 2004, supra). In order to enhance the binding of MASP-2, peptides can be synthesized so that they are flanked by two GPO triplets at each end to promote the formation of a triple helix found in native MBL protein ("GPO GPO GLR GLQ GPO GKL GPO GGP OGP O "SEQ ID NO: 25) (Wallis, R., et al., J. Biol. Chem. 279: 14065, 2004).

The MASP-2 inhibitory peptide may be derived from human H-picoline comprising the sequence "GAO GSO GEK GAO GPQ GPO GPO GKM GPK GEO GDO" (SEQ ID NO: 27) from the consensus MASP-2 binding domain in H- . Derived peptide comprising the sequence "GCO GLO GAO GDK GEA GTN GKR GER GPO GPO GKA GPO GPN GAO GEO" (SEQ ID NO: 28) from the consensus MASP-2 binding region in L- .

The MASP-2 inhibitory peptide can be derived from the C4 splitting site, for example, " LQRALEILPNRVTIKANRPFLVFI "(SEQ ID NO: 29) which is linked to the C-terminal part of antithrombin III (Glover, G. L, et al Immunol 25: 1261 (1988)).

Table 3: Exemplary MASP-2 inhibitory peptides SEQ ID NO Source SEQ ID NO: 6 Human MASP-2 protein SEQ ID NO: 8 The CUBI domain of MASP-2 (SEQ ID NO: 6 of aa 1-121) SEQ ID NO: 9 The CUBIEGF domain of MASP-2 (SEQ ID NO: 6 of aa 1-166) SEQ ID NO: 10 The CUBIEGFCUBII domain of MASP-2 (SEQ ID NO: 6 of aa 1-293) SEQ ID NO: 11 The EGF domain of MASP-2 (aa 122-166) SEQ ID NO: 12 The serine-protease domain (aa 429-671) of MASP-2 SEQ ID NO: 16 MBL binding region in MASP-2 SEQ ID NO: 3 Human MAp19 SEQ ID NO: 21 Human MBL protein SEQ ID NO: 22 OGK-X-GP, wherein "O" = hydroxyproline and "x" is a hydrophobic amino acid residue Synthesis of human MBL and human picoline Peptide consensus binding site SEQ ID NO: 23 OGKLG Human MBL core binding site SEQ ID NO: 24 GKR GLQ GPO GKL GPO G Human MBL Triplet 6-10-Proven Binding MASP-2 SEQ ID NO: 25 GPOGPOGLRGLQGPO GKLGPOGGPOGPO A human MBL triplet with GPO added to enhance the formation of a triple helix SEQ ID NO: 26 GKDGRDGTKGEKGEP GQGLRGLQGPOGKLG POGNOGPSGSOGPKG QKGDOGKS Human MBL triplet 1-17 SEQ ID NO: 27 GAOGSOGEKGAOGPQ GPO GPOGKMGPKGEO GMO Human H-picoline (Hataka) SEQ ID NO: 28 GCOGLOGAOGDKGE AGTNGKRGERGPOGP OGKAGPOPNGAOGEO Human L-picoline P35 SEQ ID NO: 29 LQRALEILPNRVTIKA NRPFLVFI Human C4 cleavage site

Note: The letter "O" represents hydroxyproline. The letter "X" is a hydrophobic residue.

The C4 cleavage site that inhibits the MASP-2 serine protease site and the peptides derived from other peptides are chemically modified to become irreversible protease inhibitors. For example, suitable modifications include adding to the C-terminal halomethylketone (Br, Cl, I, F), Asp or GIu, or functional side chains; Haloacetyl (or other? -Haloacetyl) group on the amino group or other functional side chain; An amino or carboxy terminal or functional side chain epoxide or an imine-containing group; Or imidate esters on amino or carboxy termini or functional side chains. Such modifications are useful for permanent enzyme inhibition by covalent attachment of the peptides. Whereby a low effective dosage and / or less frequent administration of the peptide inhibitor is required.

MASP-2 inhibitory peptides useful in the methods of the invention, along with the inhibitory peptides described above, include peptides containing the MASP-2-linked CDR3 region of the anti-MAASP-2 MoAb obtained as described herein. The sequence of the CDR regions used for peptide synthesis is determined by methods known in the art. The heavy chain variable region generally ranges from 100 to 150 amino acids in length. The light chain variable region generally ranges from 80 to 130 amino acids in length. The CDR sequences within the heavy and light chain variable regions include only about 3-25 amino acid sequences that are readily sequenced by those skilled in the art.

Those skilled in the art have recognized that substantially homologous mutations of the MASP-2 inhibitory peptides described above exert their MASP-2 inhibitory activity. Exemplary variations include, but are not necessarily limited to, peptides having amino acid insertions, deletions, substitutions, and / or additions at the carboxy-terminal or amino-terminal portion of the subject peptides and mixtures thereof. Thus, homologous peptides with MASP-2 inhibitory activity are considered useful in the methods of the present invention. The peptides described above may include cloning motifs and other modifications with conservative substitutions. Conservative variants are described herein and include the exchange of other amino acids such as charge, size or hydrophobicity.

MASP-2 inhibitory peptides are modified to increase solubility and / or minimize positive or negative charge to make the moieties within the intact protein more similar. Derivatives may or may not retain the exact primary amino acid structure of a peptide as long as the derivative has functionally retained the desired properties of MASP-2 inhibition. Modifications include substitution of a substituted amino acid by one of the commonly known 20 amino acids or another amino acid, an amino acid or D-amino acid derivatized or substituted to have additional desirable properties, such as resistance to enzymatic degradation, or another molecule Substitution by a carbohydrate, amino acid or peptide that mimics the natural stereostructure and function of a compound, e.g., an amino acid; Amino acid deletion; Derived amino acids or D-amino acids or other molecules or compounds which have amino acid insertions by commonly known 20 amino acids or other amino acids, with the desired additional properties, for example resistance to enzymatic degradation, such as amino acids Substitution by a carbohydrate, amino acid or peptide that mimics the natural stereostructure or function of the molecule; Or substitution by another molecule or compound such as a carbohydrate or nucleic acid monomer that mimics the natural stereostructure, charge sharing and function of the peptide. The peptide may be modified by acetylation or amidation.

The synthesis of derivative inhibition peptides may depend on known techniques such as peptide biosynthesis, carbohydrate biosynthesis, and the like. As a starting point, the skilled artisan can use a suitable computer program to determine the stereostructure of the subject peptide. Where the peptide stereostructures of the present disclosure are known, the skilled artisan will appreciate that derivatives possessing properties that are not provided or may be enhanced over those found in the peptide, but any type of substitution has a basic stereostructure and charge distribution of the peptide It can be determined within a reasonable design method whether it can be made in more than one site to form. If candidate derivative molecules are identified, the derivatives can be tested using the assay described above to determine if they function as MASP-2 inhibitors.

Screening of MASP-2 inhibitory peptides

Molecular modeling and rational molecular design can be used to generate and screen peptides that mimic the molecular structure of the core binding region of MASP-2 and inhibit complement activation of MASP-2. The molecular structure used in the modeling is important for the MASP-2 function, including the CDR region of the anti-MASP-2 monoclonal antibody and the region required for dimerization, the region involved in binding to MBL, and the serine protease activation site described above. &Lt; / RTI &gt; Peptide identification methods that bind to specific targets are known in the art. For example, molecular imprinting is used in the new production of macromolecular structures, such as peptides that bind to specific molecules. See, for example, Shea, KJ, "Molecular Imprinting of Synthetic Network Polymer: The New Synthesis of Micromolecular Binding and Catalytic Sties," TRIP 2: (5)

As an illustrative example, a method for preparing a MASP-2 binding peptide mimetic is as follows. The binding region of an anti-MASP-2 antibody that exhibits a functional monomer or MASP-2 inhibition (template) of a known MASP-2 binding peptide was polymerized. The template was then removed and the polymerization of the second class of monomers was carried out within a blank space by a template providing the molecules of the stigmata exhibiting one or more characteristics similar to the template. Other MASP-2 binding molecules, such as MASP-2 inhibitors such as polysaccharides, nucleosides, drugs, nuclear proteins, lipid proteins, carbohydrates, glycoproteins, steroids, lipids and other biologically active substances, . This method is useful for the design of a variety of biological mimics that are more stable than natural counterparts because they are produced by free radical polymerization of functional monomers which are generally compounds with non-biodegradable skeletons.

Peptide synthesis

MASP-2 inhibitory peptides may be prepared by techniques known in the art, such as those described in Merrifield, in J. Amer. Chem. Soc. 85: 2149-2154, 1963, which is incorporated herein by reference in its entirety. Automated synthesis can be accomplished, for example, by the Applied Biosystems 43 IA peptide synthesizer (Foster City, Calif.) According to the manufacturer's instructions. Other techniques can be found in, for example, Bodanszky, M., et al., Peptide Synthesis, second edition, John Wiley & Sons, 1976, and other references known in the art.

The peptides may be prepared using standard gene engineering techniques known to those skilled in the art. For example, the peptides can be enzymatically produced by insertion of a nucleic acid encoding the peptide into an expression vector, DNA expression, and DNA transfer to the peptide in the presence of the required amino acid. The peptide may be fused by insertion into an expression vector in a phase with a peptide encoding the carrier protein encoding the carrier protein and subsequently separated and purified using a carrier protein isolated from the peptide. Fusion protein-peptides can be separated using chromatography, electrophoresis, or immunological techniques (e.g., binding to the resin through an antibody of the carrier protein). Peptides can be degraded by chemical methods or enzymatically, for example, by hydrolases.

MASP-2 inhibitory peptides useful in the methods of the invention may be produced in recombinant host cells following conventional techniques. In order to express a MASP-2 inhibiting peptide encoding the sequence, the nucleic acid molecule encoding the peptide must be operably linked to a sequence that is regulated in the expression vector and then introduced into the host cell. For example, a promoter and an enhancer, an expression vector may be included in a marker gene and a translation control sequence suitable for selection of a cell carrying the expression vector.

The nucleic acid molecule encoding the MASP-2 inhibitory peptide can be synthesized as a "gene machine" using a protocol such as the phosphoramidite method. When chemically synthesized double-stranded DNA is required for the synthesis of, for example, genes or gene segments, each complementary strand is each produced. The production of short genes (60-80 basepairs) is technically direct and can be accomplished by synthesizing complementary strands and then annealing. In the production of long genes, synthetic genes (double-stranded) are assembled in a modular form in single-stranded segments of 20 to 100 nucleotides in length. For the study of polynucleotide synthesis, see, for example, Glick and Pasternak, "Molecular Biotechnology, Principles and Applications of Recombinant DNA ", ASM Press, 1994; Itakura, K., et al., Annu. Rev. Biochem. 53: 323,1984; And Climie, S., et al., Proc. Nat'l Acad. ScL USA 87: 633, 1990.

Small molecule inhibitor

In one embodiment, the MASP-2 inhibitor is a small molecule inhibitor comprising low molecular weight natural and synthetic materials such as, for example, peptides, peptidomimetics and non-peptide inhibitors, including oligonucleotides and organic compounds. The small molecule inhibitor of MASP-2 can be generated based on the molecular structure of the variable region of the anti-MASP-2 antibody. Small molecule inhibitors can be designed and generated based on MASP-2 crystal structures using computerized drug design (Kuntz LD, et al., Science 257: 1078, 1992). The crystal structure of LAT MASP-2 has been described in the literature (Feinberg, H., et al., EMBO J. 22: 2348-2359, 2003). Using the method by Kuntz et al., The MASP-2 crystal structure arrangement is used at the input of a computer program, e.g., DOCK, to produce a list of small molecule structures capable of binding to MASP-2. The use of such computer programs is well known to those skilled in the art. For example, the crystal structure of an HIV-I protease inhibitor can be used to identify the unique non-peptide ligand that is an HIV-I protease inhibitor by evaluating the compound sought in the Cambridge crystallography database using the program DOCK to fit the binding site of the enzyme (Kuntz, LD., Et al., J. Mol. Biol., 161: 269-288, 1982, DesJarlais, RL, et al., PNAS 87: 6644-6648, 1990).

A list of small molecule structures identified by computerization as potential MASP-2 inhibitors was selected, for example, using the MASP-2 binding assay described in Example 7. Small molecules known to bind to MASP-2 are measured, for example, by the functional assay of Example 2 to determine whether they inhibit MASP-2-dependent complement activation.

MASP-2 soluble receptor

Other suitable MASP-2 inhibitors appear to include MASP-2 soluble receptors and are prepared using methods known to those skilled in the art.

MASP-2 expression inhibitor

In another embodiment of the features of the invention, the MASP-2 inhibitor is a MASP-2 expression inhibitor that inhibits MASP-2-dependent complement activation. Representative MASP-2 expression inhibitors include MASP-2 antisense nucleic acid molecules (e.g., antisense mRNA, antisense DNA or antisense oligonucleotides), MASP-2 ribozyme, and MASP-2 RNAi molecules in an embodiment of the features of the invention.

Anti-sense RNA and DNA molecules directly block the translation of MASP-2 mRNA by hybridizing MASP-2 mRNA or blocking translation of MASP-2 protein. Antisense nucleic acid molecules can be prepared by a number of other methods that can interfere with the expression of MASP-2. For example, antisense nucleic acid molecules can be produced by the conversion of the coding region (or portions thereof) of the MASP-2 cDNA (SEQ ID NO: 4) relative to the anterior direction of transcription to transcribe their complement.

The antisense nucleic acid molecule generally corresponds at least substantially to a portion of the target gene or gene. The nucleic acid, however, does not have to be completely identical to inhibit expression. Generally, high homology can be used to compensate for the use of short antisense nucleic acid molecules. The minimum percentage concordance is generally about 65% or more, but the high percentage concordance can exert more effective suppression of the expression of the innate sequence. A substantially high percentage percent agreement of greater than about 80% is generally preferred, with between about 95% and full agreement generally being most preferred.

It is not necessary for the antisense nucleic acid molecule to have the same intron or exon pattern as the target gene and the non-coding portion of the target gene may equally be effective in achieving antisense suppression of target gene expression as an encoding portion. Although a long sequence is preferred, a DNA sequence of at least about eight nucleotides may be used as the antisense nucleic acid molecule. In the present invention, an exemplary useful example of an inhibitor of MASP-2 is an antisense MASP-2 nucleic acid molecule that is at least 90% identical to the complement of the MASP-2 cDNA consisting of the nucleic acid sequence of SEQ ID NO: The nucleic acid sequence of SEQ ID NO: 4 encodes a MASP-2 protein consisting of the amino acid sequence of SEQ ID NO: 5.

Targeting of antisense oligonucleotides that bind to MASP-2 mRNA is another mechanism used to reduce the level of MASP-2 protein synthesis. For example, the synthesis of polygalactononase and muscarinic type 2 acetylcholine receptors is inhibited by antisense oligonucleotides related to their respective mRNA sequences (US Patent No. 5,739,119, Cheng, and US Patent No. 5,131, 242). No. 5,759,829, Shewmaker). In addition, examples of antisense inhibition have been demonstrated with nuclear protein cyclin, multidrug inhibitory gene (MDGl), ICAM-1, E-celectin, STK-I, striated GABA _A receptor and human EGF. 5,801,154, Baracchini, US Patent No. 5,789,573, Baker, US Patent No. 5,718,709, Considine, and US Patent No. 5,610,288, Reubenstein).

The system is used by those skilled in the art to determine whether oligonucleotides on probes for suitable sites in target mRNA are useful for the present invention using RNAse H cleavage as an index of sequence accessibility within a transcription. Scherr, M., et al, Nuclear Acids Res. A mixture of antisense oligonucleotides complementary to a specific region of the MASP-2 transcription was used to express MASP-2 (Moss et al. Cell extracts, such as hepatocytes, and hybridized to produce vulnerable regions of RNAse H. This method can be used to produce dimers, hairpins, or other 2 &lt; RTI ID = 0.0 &gt; Can be combined with computer-assisted sequence selection that anticipates optimal sequence selection of antisense compositions based on their ability to form secondary structures. Such secondary structure analysis and target site selection considerations may be combined with oligo primer analysis software (Rychlik, L, 1997) and BLASTN 2.0.5 algorithm software (Altschul, SF, et al, Nucl. Acids Res. 25: 3389-3402, 1997). The antisense oligonucleotides comprising about 9 to about 35 nucleotides are particularly preferred. The inventors have found that all oligonucleotide compositions in the range of 9 to 35 nucleotides (I. E., 9,10,11,12,13,14,15,16,17,18,19,20,21,22,23,24,25,26,27,28,29,30,31,32 , 33, 34, or 35 base lengths) is highly desirable for the practice of the antisense oligonucleotide-based methods of the present invention. The highly preferred MASP-2 mRNA target region is the AUG translation initiation codon, (SEQ ID NO: 4). Exemplary MASP-2 expression inhibitors are shown in Table 4 as being substantially complementary to the region of the MASP-2 gene, for example, between the -10 and +10 regions of the MASP-2 gene nucleotide sequence .

Table 4: Exemplary expression inhibitors of MASP-2 SEQ ID NO: 30 (nucleotides 22-680 of SEQ ID NO: 4) The nucleotide sequence (SEQ ID NO: 4) of the CUBIEGF-encoding MASP-2 cDNA SEQ ID NO: 31 5'CGGGCACACCATGAGGCTGCTG ACCCTCCTGGGC3 ' Nucleotides 12-45 of SEQ ID NO: 4 comprising the MASP-2 translation initiation site (sense) SEQ ID NO: 32 5'GACATTACCTTCCGCTCCGACTC CAACGAGAA3 ' Nucleotides 361-396 of SEQ ID NO: 4 which encode a region comprising the MASP-2 MBL binding site (sense) SEQ ID NO: 33 5 'AGCAGCCCTGAATACCCACGGCC GTATCCCAAA3' Nucleotides 610-642 of SEQ ID NO: 4 encoding the region containing the CUBII domain capability

As used herein before, the term "oligonucleotide" as used herein refers to ribonucleic acid (RNA) or deoxyribonucleic acid (DNA) or their parent oligomer or polymer. The term includes oligonucleotide bases composed of oligonucleotides having naturally occurring nucleotides, shared (skeletal) linkages between sugars and nucleosides, and non-naturally occurring strains. Such modifications allow the introduction of naturally occurring oligonucleotides, such as reduced toxicity, increased stability to nuclease degradation, and any desirable properties not provided through increased cellular aspiration. In an exemplary embodiment, the antisense compounds of the present invention differ from native DNA in that the phosphate substituent modifies the phosphodiester backbone to prolong the survival of the antisense oligonucleotides replaced by phosphorothioate. As such, one or both ends of the oligonucleotide may be replaced by one or more acridine derivatives interposed between adjacent base pairs in the nucleic acid strand.

Another alternative to antisense is to use "RNA interference" (RNAi). Double-stranded RNAs (dsRNAs) can induce gene splicing in mammals in vivo. The natural function and sub-inhibition of RNAi is likely to protect the genome against invasion by transgenic elements such as retrotransposons and, if activated, viruses producing variant RNA or dsRNA in host cells (see, e.g., Jensen, J. et al. , et al., Nat Genet., 21: 209-12, 1999). Double-stranded RNA molecules can be prepared by synthesizing two RNA strands, each of which can form a double-stranded RNA molecule, each about 19-25 nucleotides in length (e.g., 19-23 nucleotides). For example, dsRNA molecules useful in the methods of the invention may comprise RNA corresponding to a sequence, and their complement are listed in Table 4. Preferably, at least one RNA strand has a 3 ' overhang at 1-5 nucleotides. The synthesized RNA strands are bound under the condition of forming double-stranded molecules. The RNA sequence comprises at least 8 nucleotide portions of SEQ ID NO: 4 with a total length of 25 nucleotides or less. The design of siRNA sequences of a given target is within the skill of the art. Commercial services can be used for siRNA sequence design, guaranteeing at least 70% knockdown of expression (Qiagen, Valencia, Calif).

The dsRNA is administered as a pharmaceutical composition and is carried out by known methods, wherein the nucleic acid is introduced into the desired target cell. Commonly used gene transfer methods include calcium phosphate, DEAE-dextran, electroporation, microinjection and viral methods. Such methods have been disclosed in Ausubel et al., Current Protocols in Molecular Biology, John Wiley & Sons, Inc., 1993.

A ribozyme can be used to reduce the amount and / or biological activity of MASP-2, for example, a ribozyme that targets MASP-2 mRNA. A ribozyme is a catalytic RNA molecule capable of cleaving a nucleic acid molecule having a sequence completely or partially homologous to the sequence of a ribozyme. The ribozyme transfer gene design allows the RNA ribozyme to specifically compete with the target RNA to degrade the phosphodiester backbone at a specific site and thus functionally inactivate the target RNA. In the performance of this cleavage, the ribozyme is not transformed per se and is therefore recycled to enable decomposition of other molecules. A ribozyme sequence is included in the antisense RNA to confer RNA-cleaving activity on them. Thus increasing the activity of the antisense construct.

A ribozyme useful in the practice of the present invention generally comprises a hybridization region of at least about 9 nucleotides that is complementary to at least a portion of the target MASP-2 mRNA within the nucleotide sequence and is catalytically active to divide the target MASP-2 mRNA (Generally EPA No. 0321 201; WO 88/04300; Haseloff, J., et al, Nature 334: 585-591, 1988; Fedor, MJ, et al., Proc. Natl. Acad. ScL USA 87: 1668-1672, 1990; Cech, TR, et al., Ann. Rev. Biochem. 55: 599-629, 1986).

A ribozyme can be directly targeted to a cell in the form of an RNA oligonucleotide comprising a ribozyme sequence or can be introduced into a cell as an expression vector encoding the desired ribozyme RNA. The ribozyme can be used or applied in the same manner as described in the antisense polynucleotide.

The anti-sense RNA and DNA, ribozyme and RNAi molecules used in the method of the present invention can be prepared by synthesizing all known DNA and RNA molecules. This includes chemical synthesis techniques of known oligodeoxyribonucleotides and oligoribonucleotides such as, for example, solid phase phosphoramidite chemical synthesis. Alternatively, the RNA molecule can be generated by in vitro and in vivo transcription of a DNA sequence encoding an antisense RNA molecule. Such DNA sequences may be integrated into a variety of vectors integrated into suitable RNA polymerase promoters, such as T7 or SP6 polymerase promoters. Alternatively, antisense cDNA constructs encoding the antisense RNA structurally or inducibly, depending on the promoter used, can be stably introduced into the cell line.

A variety of known DNA molecule modifications can be introduced as a means of increasing stability and half-life. Useful modifications include the addition of a side-protecting sequence of the ribonucleotide or deoxyribonucleotide to the 5 ' and / or 3 ' end of the molecule or the addition of a phosphorothioate or 2 ' O -Methyl, &lt; / RTI &gt;

V. Pharmaceutical Compositions and Methods of Delivery

In another aspect, the invention provides a composition for inhibiting the adverse effects of MASP-2-dependent complement activation comprising a therapeutically effective amount of a MASP-2 inhibitor and a pharmaceutically acceptable carrier. MASP-2 inhibitors are administered to a subject in need thereof in therapeutically effective doses to treat or ameliorate MASP-2-dependent complement-activating afferents. A therapeutically effective dose refers to an amount that improves symptoms as a sufficient amount of a MASP-2 inhibitor.

The toxicity and therapeutic efficacy of the MASP-2 inhibitor was determined by standard pharmaceutical procedures using an experimental animal model, e.g., a murine MASP-2 - / - mouse model expressing the human MASP-2 insert gene of Example 3. Using these animal models, NOAEL (no observed adverse effect levels) and MED (minimum effect dose) can be determined by standard methods. The dose rate between the NOAEL and MED effects is the therapeutic ratio, expressed as the NOAEL / MED ratio. MASP-2 inhibitors that exhibit large therapeutic ratios or indicators are most preferred. The data obtained from cell culture assays and animal studies are used to formulate a range of doses used in humans. The dosage of the MASP-2 inhibitor is preferably in the range of circulating concentrations that include MED with little or no toxicity. Dosages may vary within this range depending on the dosage form employed and the route of administration.

For all compound formulations, the therapeutically effective dose was assessed using animal models. For example, dosages may be formulated in animal models to achieve a circulating plasma concentration range that includes MED. Quantitative levels of MASP-2 inhibitors in plasma can be measured, for example, by high performance liquid chromatography.

In conjunction with toxicity studies, effective dosages can be assessed based on the amount of MASP-2 protein in a living subject and the binding affinity of a MASP-2 inhibitor. MASP-2 levels in normal human subjects are present in low levels of serum in the range of 500 ng / ml, and levels of MASP-2 in certain subjects may be determined using quantitative assays of MASP-2, -Kristensen M., et al., J. Immunol. Methods 282: 159-167, 2003.

In general, the dosage of a composition comprising a MASP-2 inhibitor will depend on such factors as the age, weight, height, sex, general medical symptoms, and previous medical history of the subject. By way of example, a MASP-2 inhibitor such as an anti-MASP-2 antibody can be administered at a subject body weight of about 0.010 to 10.0 mg / kg, preferably 0.010 to 1.0 mg / kg, more preferably 0.010 to 0.1 mg / kg have. In one embodiment, the composition may comprise a combination of an anti-MAST-2 antibody and a MASP-2 inhibitory peptide.

The therapeutic efficacy, and the appropriate dosage, of a MASP-2 inhibitory composition and a method of the invention in a given subject can be determined according to complement assays known to those of skill in the art. Complementation produces a variety of specific products. Over the last decade, sensitive and specific assays have been developed and most of these activation products such as small activation fragments C3a, C4a, and C5a and large activation fragments iC3b, C4d, Bb, and sC5b-9 are available . Most of these assays utilize monoclonal antibodies that react with new antigens (new antigens) exposed on non-existing fragments of the protein. Where they are formed and make this measurement very simple and specific. Most depend on ELISA technology, but radioimmunoassays are also used for C3a and C5a. This latter measure measures both untreated sections and their 'desArg' sections, which are the major forms found in the circulation. Untreated slices and C5a _deSArg bind rapidly to cell surface receptors and are rapidly removed and present at very low concentrations while C3a _deSArg is not bound to cells and accumulates in plasma. Measurement of C3a provides a sensitive, pathway-independent, complement activation indicator. Alternate pathway activation can be assessed by measuring the Bb intercept. Detection of the fluid-phase product, sC5b-9, of the membrane attack pathway provides evidence that the complement is active and complete. Since both lectins and classical pathways produce the same activation products, C4a and C4d, measurement of these two fragments does not provide information that these two pathways produce an activated product.

Additional medications

The compositions and methods comprising a MASP-2 inhibitor may optionally comprise one or more additional therapeutic agents, which increase the activity of the MASP-2 inhibitor or provide an adjunctive therapeutic function in an additional or synergistic manner. For example, one or more MASP-2 inhibitors may be administered in combination with one or more anti-inflammatory and / or analgesic agents. The inclusion and selection of additional agent (s) will be determined to achieve the desired therapeutic result. Suitable anti-inflammatory and / or analgesic agents include: serotonin receptor antagonists; Serotonin receptor agonists; Histamine receptor antagonists; Bradykinin receptor antagonists; Calicaine inhibitors; Tachykinin receptor antagonists, such as neurokinin and neurokinin 2 receptor subtype antagonists; Calcitonin gene-linked peptide (CGRP) receptor antagonists; Interleukin receptor antagonists; (COX-1, COX-2, or non-selective) inhibitors of enzymatic activity in the arachidonic acid metabolite synthesis pathway, including, for example, PLA2 islet inhibitors and PLCγ islet inhibitors, cyclooxygenase COX-1 and -2 inhibitors), lipoxygenase inhibitors; Prostanoid receptor antagonists such as eniconoside EP-1 and EP-4 receptor subtype antagonists and thromboxane receptor subtype antagonists; Leukotriene receptor antagonists such as leukotriene B4 receptor subtype antagonists and leukotriene D4 receptor subtype antagonists; Opioid receptor agonists such as mu-opioids, delta-opioids, and kappa-opioid receptor subtype agonists; Purinoseceptor agonists and antagonists such as P2X receptor antagonists and P2Y receptor agonists; Adenosine triphosphate (ATP) -sensitive potassium channel openers; MAP kinase inhibitors; Nicotinic acetylcholine inhibitors; And alpha adrenergic receptor agonists (including alpha-1, alpha-2, and non-selective alpha-1 and 2 agonists).

When used in the prevention or treatment of restenosis, the MASP-2 inhibitors of the present invention may be combined with one or more anti-restenosis agents for simultaneous administration. Suitable anti-restenotic agents include: antiplatelet agents such as: thrombin inhibitors and receptor antagonists, adenosine diphosphate (ADP) receptor antagonists (purinoseceptor 1 receptor antagonists), thromboxane inhibitors and receptor antagonists and platelet membrane glycoprotein receptor antagonists; Inhibitors of cell adhesion molecules, such as cerectin inhibitors and integrin inhibitors; Anti-coagulant; Interleukin receptor antagonists; And intracellular signaling inhibitors such as: protein kinase C (PKC) inhibitors and protein tyrosine phosphatases, modulators of intracellular protein tyrosine kinase inhibitors, inhibitors of the src homologous 2 (SH2) domain, and calcium channel antagonists.

The MASP-2 inhibitors of the present invention may be administered in combination with one or more other complement inhibitors. There is no complement inhibitor currently approved for humans, but some pharmacological agents seem to block the complement in vivo. Many of these agents are toxic or only partial inhibitors (Asghar, S. S., Pharmacol. Rev. 36: 223-44, 1984) and their use is limited as a research tool. K76COOH and nafamstat mesilate have been shown to be partially effective in animal implant models (Miyagawa, S., et al., Transplant Proc. 24: 483-484, 1992). Low molecular weight hairpins appear to be effective in regulating complement activation (Edens, R. E., et al., Complement Today, pp. 96-120, Basel: Karger, 1993). It is believed that such small molecule inhibitors are useful agents for the combined use of the MASP-2 inhibitors of the present invention.

Other naturally occurring complement inhibitors may be used in combination with the MASP-2 inhibitors of the present invention. Biologic inhibitors of complement include soluble complement factor 1 (sCR1). It is a naturally occurring inhibitor and can be found on the outer membrane of human cells. Other membrane inhibitors include DAF, MCP, and CD59. Recombinant forms were shown for their in vitro and in vivo anti-complement activation. sCR1 appears to be effective for xenotransplantation, where the complement system (both replacement and classical) triggers the hypersensitive reactive rejection syndrome in the blood perfusion between the freshly transplanted organs (Platt JL, et al., Immunol. Today 77: 450-6, 1990 Marino IR, et al., Transplant Proc 1071: 6, 1990; Johnstone, PS, et al., Transplant 54: 573-6, 1992). The use of sCR1 protects the survival time of transplanted organs and is associated with the complement pathway in the pathogenesis of long-term survival (Leventhal, JR et al., Transplant 55: 857-66, 1993; Pruitt, SK, et al., Trnasplant 57: 363-70, 1994).

Additional complement inhibitors suitable for use in combination with the compositions of the present invention also include those being developed by MoAbs, such as Alexion Pharmaceuticals, Inc. (New Haven, Connecticut), and anti-proPeridine MoAbs.

When used in the treatment of arthritis (e.g., osteoarthritis and rheumatoid arthritis), the MASP-2 inhibitors of the present invention may be combined with one or more cartilage protecting agents, which include one or more cartilage stimulating promoters and / or one or more cartilage inhibitors, May include both the anchoring agent and the cryoprotectant at the same time. Suitable anabolic cartilage protectants include, but are not limited to, interleukin (IL) receptor agonists such as IL-4, IL-IO, IL-13, rhIL-4, rhIL-10 and rhIL-13, and chimeric IL- IL-13; BMP-2, BMP-4, BMP-5, BMP-6, BMP-1, TGF-beta1, TGF- -7 (OP-1), and OP-2 / BMP-8, growth-differentiating factors such as GDF-5, GDF-6 and GDF-7, recombinant TGF-? S and BMPs and chimeric TGF-? S and BMPs; Insulin-like growth factors such as IGF-1; And fibroblast growth factors such as bFGF. Suitable cholinergic cartilage protectants include, but are not limited to, interleukin-1 (IL-I) receptor antagonists (IL-1ra), such as the soluble human IL-I receptor (shuIL-lR), rshuIL-lR, rhlL- , And AF12198; A soluble receptor, a recombinant TNF soluble receptor, and a chimeric TNF soluble receptor such as a chimeric rhTNFR: Fc, Fc fusion soluble receptor and an anti-TNF antibody comprising a tumor necrosis factor (TNF) receptor antagonist (TNF-a) such as sTNFRl and sTNFRII; (COX-2 specific) inhibitors such as DuP 697, SC-58451, celecoxib, rofecoxib, nimesulide, diclofenac, meloxicam, piroxicam, NS-398, RS-57067, SC-57666, SC- , flosulide, etodolac, L-745,337 and DFU-T-614; Inhibitors of mitogen-activated protein kinase (MAPK) inhibitors such as ERK1, ERK2, SAPK1, SAPK2a, SAPK2b, SAPK2d, SAPK3 such as SB 203580, SB 203580 iodo, SB202190, SB 242235, SB 220025, RWJ 67657, RWJ 68354, FR 133605, L-167307, PD 98059, PD 169316; Inhibitors of the nuclear factor kappa B (NFKB), such as phenylethyl caffeate (CAPE), DM-CAPE, SN-50 peptide, hymenialdehyde and pyrrolidon dithiocarbamate; (NOS) inhibitors such as N ^G -monomethyl-L-arginine, 1400 ^W , diphenylenediiodinium, S-methylisothiourea, S- (aminoethyl) isothiourea, LN ⁶ - -Iminoethyl) lysine, 1,3-PBITU, 2-ethyl-2-thiopseudourea, aminoguanidine, N ^? -Nitro-L-arginine and N ^? MMP-I, MMP-12, MMP-13, MMP-7, MMP-8, MMP-9, MMP-IO, MMP- , MMP-14 and MMP-15, and inhibitors of U-24522, minocycline, 4-Abz-Gly-Pro-D-Leu-D-AIa-NHOH, Ac- NHi, rhuman TIMP1, Ryuman TIMP2, and phosphoramidon; Integrin agonists and antagonists including cell adhesion molecules such as [alpha] v [beta] 3 MoAb LM 609 and echistatin; Anti-coagulants such as F-Met-Leu-Phe receptor, IL-8 receptor, MCP-1 receptor and MIP1-I / RANTES receptor; (I) a protein kinase C (PKC) inhibitor (isozyme) comprising calpostin C, G-6203 and GF 109203X rolls, and (ii) a protein kinase inhibitor such as a protein tyrosine kinase Inhibitors; (b) modulators of intracellular protein tyrosine phosphatases (PTPases); And (c) an SH2 domain (src homologous 2 domain) inhibitor.

In some applications, it is beneficial to administer the MASP-2 inhibitor of the present invention in combination with a spasm inhibitor. For example, in urographic applications, it is advantageous to include at least one smooth muscle spasm suppressor and / or at least one anti-inflammatory agent, wherein at least one vasospasm inhibitor and / or at least one anti- It is useful to include anti-inflammatory agents and / or at least one anti-restenotic agent. Suitable examples of spasm inhibitors include: serotonin 2 receptor subtype antagonists; Tachykinin receptor antagonists; Nitric oxide donors; ATP-sensitive potassium channel openers; Calcium channel antagonists; And endothelin receptor antagonists.

Pharmaceutical carriers and delivery vehicles

In general, the MASP-2 inhibitor compositions of the present invention in combination with other selected therapeutic agents are suitably included in a pharmaceutically acceptable carrier. The carrier is chosen to be non-toxic, biocompatible, and not critically affecting the biological activity of the MASP-2 inhibitor (and any other therapeutic agent associated therewith). Exemplary pharmaceutically acceptable carriers for peptides are described in U.S. Pat. Patent No. 5,211, 657, Yamada. Anti-MASP-2 antibodies and inhibitory peptides useful in the present invention may be administered in solid, semi-solid, gel, liquid or gaseous forms such as tablets, capsules, powders, granules, ointments, solutions, suppositories, Can be formulated into possible scans. The present invention also enables local administration of the compositions by coating medical devices and the like.

Suitable carriers for parenteral and topical delivery via injection, infusion or perfusion include distilled water, physiological phosphate-buffered saline, normal or lactated Ringer's solution, dextrose solution, Hank's solution, or propanediol. In addition, sterile, fixed oils may be used as a solvent or suspension medium. For this purpose, all biocompatible oils may be used including synthetic mono- or diglycerides. Along with this, fatty acids such as oleic acid are used in injectable products. The carriers and formulations may be formulated as liquids, suspensions, polymerizable or non-polymerizable gels, pastes or ointments.

The carrier also includes a delivery vehicle that sustains (i.e., prolongs, slows, or modulates) the delivery of the agent (s) or promotes delivery, inhalation, stability, or pharmacokinetics of the agent (s). Such delivery vehicles include, as non-limiting examples, microparticles, microspheres, nanospheres or nanoparticles composed of proteins, liposomes, carbohydrates, synthetic organic compounds, inorganic compounds, polymeric or copolymeric hydrogels and polymeric micelles . Suitable hydrogel and micelle delivery systems include PEO: PHB: PEO copolymers and copolymers / cyclodextrin complexes (WO 2004/009664 A2) and PEO and PEO / cyclodextrin complexes (US 2002/0019369 A1). Such hydrogels may be injected locally at the site of the intended activity, or may be injected subcutaneously or intramuscularly to form a sustained-release reservoir. For intra-articular delivery, the MASP-2 inhibitor may be carried in an injectable liquid or gel carrier as described above, and may be carried on the aforementioned injectable sustained-release delivery carrier, or hyaluronic acid or hyaluronic acid derivative.

For oral administration of a non-peptidic agent, the MASP-2 inhibitor may be carried in an inert filler or diluent such as sucrose, corn starch, or cellulose.

For topical administration, the MASP-2 inhibitor is loaded onto the microcapsule delivery system via ointment, lotion, cream, gel, drop, suppository, spray, liquid or powder, or gel or transdermal patch.

Various nasal and pulmonary delivery systems, such as aerosols, metered dose inhalers, dry powder inhalers, and atomizers, are under development and may be suitably adapted for delivery of the invention in an aerosol, inhaler, or spray delivery carrier, respectively.

A suitable sterile delivery system (e.g., liquid; gel, suspension, etc.) may be used for administration of the present invention for intrathecal (IT) or intracerebroventricular (ICV) delivery.

The compositions of the present invention may also include biocompatible excipients such as dispersing or wetting agents, suspending agents, diluents, buffers, absorption enhancers, emulsifiers, binders, thickeners, flavoring agents (for oral administration) and the like.

Pharmaceutical carriers for antibodies and peptides

With respect to anti-MAST-2 antibodies and inhibitory peptides, an exemplary formulation comprises a solution or suspension of a compound in a physiologically acceptable diluent with a pharmaceutical carrier, which may be a sterile liquid such as water, oil, saline, glycerol or ethanol May be administered parenterally as an injectable dose. In addition, adjuvants such as wetting or emulsifying agents, surfactants, pH buffering substances and the like may be present in the composition comprising the anti-MAST-2 antibody and the inhibitory peptide. Additional components of the pharmaceutical composition include petroleum (e.g., animal, plant or synthetic origin), for example, soybean oil and mineral oil. Generally, glycols such as propylene glycol or polyethylene glycol are preferred liquid carriers for injectable solutions.

Anti-MAST-2 antibodies and inhibitory peptides may be administered in the form of a storage injection or implant product that may be formulated in this manner to allow for sustained or pulsatile release of the active agent.

A pharmaceutically acceptable carrier for an expression inhibitor

More particularly, with respect to expression inhibitors useful in the methods of the present invention, the compositions are provided comprising the aforementioned expression inhibitors and pharmaceutically acceptable carriers or diluents. The composition may further comprise a colloidal dispersion system.

Pharmaceutical compositions comprising an expression inhibitor include, but are not limited to, solutions, emulsions, and liposome-containing formulations. Such compositions can be made from a variety of ingredients including, but not limited to, preformed liquids, self-emulsified solids and self-emulsifying solids. The products of such compositions generally relate to the combination of an expression inhibitor and one or more of the following: buffers, antioxidants, low molecular weight polypeptides, proteins, amino acids, carbohydrates such as glucose, sucrose or dextrin, chelating agents such as EDTA, glutathione And other stabilizers and excipients. Neutral buffered saline or saline mixed with non-specific serum albumin is an example of a suitable diluent.

In one embodiment, the composition can be prepared and formulated as an emulsion, which is a heterogeneous system of liquid, typically dispersed in the form of droplets (Idson, in Pharmaceutical Dosage Forms, Vol. 1, Rieger and Banker Dekker, Inc., NY, 1988). Examples of naturally occurring emulsifiers used in emulsion formulations include acacia, beeswax, lanolin, lecithin and phosphatide.

In one embodiment of the invention, the composition comprising the nucleic acid can be formulated into a microemulsion. As used herein, microemulsions refer to water, oil, and amphiphilic systems, which are single optically isotropic and thermodynamically stable liquid solutions (Rosoff in Pharmaceutical Dosage Forms, Vol. 1). The methods of the present invention can use liposomes to transfer and transfer antisense oligonucleotides to desired sites.

Pharmaceutical compositions and formulations of topical administration inhibitors can include transdermal patches, ointments, lotions, creams, gels, drops, suppositories, sprays, liquids and powders. Conventional pharmaceutical carriers, and aqueous, powder or oil based and thickening agents may be used.

Method of administration

A pharmaceutical composition comprising a MASP-2 inhibitor may be administered in a number of ways depending on whether the local or systemic administration method is most appropriate for the treatment of the condition. In addition, with respect to the extracorporeal reperfusion process of the present application, the MASP-2 inhibitor may be administered through introduction of the composition of the present invention into recirculating blood or plasma. In addition, the compositions of the present invention may be achieved by coating or including the composition on or within the implantable medical device.

Whole body transmission

As used herein, the terms "systemic delivery" and "systemic administration" include oral and parenteral routes such as intramuscular (IM), subcutaneous, intravenous (IV), arterial, inhalation, sublingual, Nasal, rectal, vaginal, and other routes of administration in which the agent delivered to a single or multiple site of intended therapeutic activity is effectively dispersed. Systemic delivery of a preferred route of the composition includes intravenous, intramuscular, subcutaneous and inhalation. The exact systemic route of administration of the selected agent using a particular composition of the invention is determined in part by the drug susceptibility to metabolic transformation pathways associated with the route of administration. For example, the peptide agent is best administered by a route other than oral route.

MASP-2 inhibitory antibodies and polypeptides may be delivered into a subject in need thereof by any suitable means. Methods for delivery of MASP-2 antibodies and polypeptides include, but are not limited to, oral, pulmonary, parenteral (e.g. intramuscular, intraperitoneal, intravenous (IV) or subcutaneous injection), inhalation (e.g. via micropowder formulations), transdermal, , Rectal, or sublingual route, and may be formulated at an appropriate dosage for each route of administration.

As a representative example, MASP-2 inhibitory antibodies and peptides can be introduced in vivo by application to body membranes capable of absorbing polypeptides, such as the nasal, gastrointestinal, and rectal membranes. The polypeptide is generally applied to the absorbent membrane in conjunction with a permeation enhancer (e.g., Lee, VHL, Crit. Rev. Ther. Drug Carrier Sys. 5:69, 1988; Lee, VHL, J. controlled release 13: ; Lee, VHL, Ed., Peptide and Protein Drug Delivery, Marcel Dekker, New York (1991); DeBoer, AG, et al., J. Controlled release, 13: 241, 1990.). For example, STDHF is a synthetic derivative of fusidic acid, structurally similar to bile salts, and has been used as a permeation enhancer for nasal delivery. (Lee, W.A., Biopharm. 22, Nov./Dec. 1990.)

MASP-2 inhibitory antibodies and polypeptides may be introduced with another molecule, such as a lipid, to protect the polypeptide from enzymatic degradation. For example, covalent attachment of polymers, particularly polyethylene glycol (PEG), has been used to protect specific proteins from enzymatic hydrolysis in the body, thus increasing half-life (Fuertges, F., et al., J Controlled release 11: 139, 1990). A number of polymer systems have been reported for protein delivery (Bae, YH, et al, J. Controlled release 9: 271, 1989; Hori, R., et al, Pharm. Res. 6: 813, 1989; Yamakawa, L J. Controlled release 10: 195, 1989; Asano, M., et al., J. Controlled release 9: J., Controlled release 9: 195, 1989; Makino, K., J. Controlled release 12: 235, 1990; Takakura, Y., et al., J. Pharm. Sci. 78: 117, 1989; Takakura, Y., et al., J. Pharm. Sci. 78: 219, 1989).

Recently, liposomes have been developed to have improved serum stability and circulating half-life (see, e.g., U.S. Patent No. 5,741,516, Webb). Additionally, liposomes and liposome-like products in a variety of ways have been studied as potential drug carriers (see, for example, US Patent No. 5,567,434, Szoka; US Patent No. 5,552,157; Yagi; US Patent No. 5,565,213; Nakamori; 5,738,868, Shinkarenko, and US Patent No. 5,795,587, Gao).

For percutaneous application, the MASP-2 inhibitory antibodies and polypeptides may be combined with other suitable components, such as carriers and / or adjuvants. The properties of these other ingredients are not limited, but they should be pharmaceutically acceptable for the intended administration and can not deteriorate the activity of the active ingredients of the composition. Examples of suitable carriers include ointments, creams, gels, or suspensions, with or without purified collagen. MASP-2 inhibitory antibodies and polypeptides can be packaged into transdermal patches, lozenges, and bandages, preferably in liquid or semi-liquid form.

The compositions of the present invention may be administered systemically at regular intervals over a determined time interval to maintain the desired level of therapeutic effect. For example, the compositions may be administered, for example, by subcutaneous injection, every 2 to 4 weeks or at less frequent intervals. The dosage regimen will be determined by the clinician taking into account various factors that may affect the combined activity of the drug. These factors include the progression of symptoms to be treated, the age, sex and weight of the patient, and other clinical factors. The dosage of the individual agent will vary depending on the function of the MASP-2 inhibitor contained in the composition and the presence and characteristics of any pharmaceutical delivery vehicle (e.g., sustained release delivery vehicle). Together, the dosage can be controlled by the frequency of administration and the pharmacokinetic behavior of the delivered drug (s).

Local delivery

As used herein, the term "localized" encompasses the application of a drug in or around an intended local active site, including, for example, local delivery, ocular delivery, intratracheal ), Intracerebroventricular (ICV), intraocular, intraocular, intracranial, or intravascular administration, placement or perfusion. Local administration can be administered at low dosages to avoid systemic side effects and to more accurately control the delivery time and concentration of the active agent at the local delivery site. Local administration provides a known concentration at the target site and is independent of the difference in metabolic blood flow in the patient. Lt; RTI ID = 0.0 &gt; direct &lt; / RTI &gt; delivery method.

Local delivery of a MASP-2 inhibitor may be accomplished in the sense of surgical treatment of a disease or condition, such as, for example, during surgery, such as arterial bypass surgery, atherectomy, laser surgery, ultrasound, balloon angioplasty and stent placement . For example, MASP-2 inhibitors may be administered to a subject during balloon angioplasty. Balloon angioplasty is the insertion of a catheter with a deflated balloon into the artery. This constricted balloon is placed around the atherosclerotic plaque and inflated to compress the plaque into the vessel wall. As a result, the balloon surface comes into contact with the vascular endothelial cells on the blood vessel surface. The MASP-2 inhibitor is attached to the balloon angioplasty catheter and is capable of releasing the drug to the atherosclerotic plaque site. The medicament may be attached to the balloon catheter according to known standard methods. For example, the medicament may be stored in the compartment of the balloon catheter until the balloon is inflated, where it is released into the local environment. Alternatively, the medicament may fill the balloon surface and contact the cells of the arterial wall when the balloon is inflated. The medicament may be delivered in a perforated balloon catheter and is described, for example, in the document Flugelman, M.Y., et al, Circulation 85: 1110-1117, 1992. PCT application WO 95/23161 discloses an exemplary method for binding therapeutic proteins to a balloon angiogenesis catheter. As such, the MASP-2 inhibitor may be included in a gel or polymeric coating applied to the stent, or may be incorporated into the material of the stent, so that the stent elutes the MASP-2 inhibitor after vascular occlusion.

MASP-2 inhibitory compositions used in the treatment of arthritis and other musculoskeletal disorders can be delivered locally by intra-articular injection. Such compositions may suitably comprise a sustained release delivery vehicle. As a further example of the need for local delivery, the MASP-2 inhibitory composition used in the treatment of the urogenital condition may suitably be placed in the bladder or in other urogenital structures.

Cloth of Medical Equipment

MASP-2 inhibitors such as antibodies and inhibitory peptides may be immobilized on (or within) the surface of an implantable or attachable medical device. The deformed surface typically comes into contact with living tissue after implantation into the animal's body. "Implantable or attachable medical device" means any device (eg, a stent and implantable drug delivery device) that is implanted or attached to an animal body tissue during normal operation. Such implantable or attachable medical devices include, for example, nitrocellulose, diazo cellulose, glass, polystyrene, polyvinyl chloride, polypropylene, polyethylene, dextran, sepharose, agar, starch, nylon, And biodegradable and / or biocompatible polymers. Binding of the protein to the device can be accomplished by techniques that do not destroy the biological activity of the bound protein, for example, by attaching one or both of the N-C-terminal residues of the protein to the device. Attachment is made at one or more internal sites within the protein. Multiple attachments (both internal or terminal of the protein) can be used. The surface of the implantable or attachable medical device may be coated with functional groups for protein fixation such as carboxyl, amide, amino, ether, hydroxyl, cyano, nitrilo, sulfanamido, acetylinic, epoxide, silanic, Anhydrous, anhydrous, crude, azido). Coupling chemistry includes, but is not limited to, formation of other bonds to functional groups utilized on ester, ether, amide, azido and sulfanamido derivatives, cyanites and MASP-2 antibodies or inhibitory peptides. MASP-2 antibodies or inhibitory fragments can be prepared by attaching affinity tag sequences to proteins such as GST (DB Smith and KS Johnson, Gene 67:31, 1988), polyhistidine (E. Hochuli et al., J. Chromatog. 411: 1987), or by addition to biotin. This affinity tag can be used to reversibly attach the protein to the device.

Proteins can be covalently attached to the surface of the device body, for example, by shared activation of the surface of the device. In a representative embodiment, the substrate cellular protein (s) can be attached to the device body by all subsequent pairs of reactors (one member of the pair present on the surface of the device body and the substrate cellular protein (s) Other member of the pair present in): hydroxyl / carboxylic acid for ester linkage; Hydroxyl / anhydride for ester linkage; Hydroxyl / isocyanate for urethane bonding. The surface of the device body, which does not have a useful reactor, can be treated with radio-frequency-released plasma (RFGD) to form a reactor that enables attachment of the substrate cellular protein (s) (e.g., Oxygen plasma therapy for the treatment of propylamino plasma for the introduction of amine groups.

MASP-2 inhibitors, including nucleic acid molecules such as antisense, RNAi- or DNA-encoding peptide inhibitors, can be inserted into a porous substrate attached to the body of the device. Representative porous substrates useful for forming the surface layer are those made from tendon or skin collagen and can be obtained from a variety of commercial sources (e.g., Sigma and Collagen Corporation), or collagen substrates may be obtained from U.S. Pat. Patent No. 4,394,370, Jefferies and 4,975,527. Was prepared as described in Koezuka. The daily collagenous material was named UltraFiber ™ and Norian Corp. (Mountain View, Calif.).

Certain polymeric substrates may be used where desired, including acrylic ester polymers and lactic acid polymers, such as those described in U.S. Pat. Patent Nos. 4,526,909, Urist, and 4,563,489, Urist. Specific examples of useful polymers include polymers of orthoesters, anhydrides, propylene-co-fumarate, or one or more a-hydroxycarboxylic acid monomers such as a-hydroxyacetic acid (glycolic acid) and / - hydroxypropionic acid (lactic acid)).

Therapy

In prophylactic applications, the pharmaceutical composition is administered to a subject that is susceptible to, or at risk for, a symptom associated with MASP-2-dependent complement activation in an amount sufficient to eliminate or reduce the risk of developing a symptom of the disease. In a therapeutic application, the pharmaceutical composition is administered to a subject suspected of having or suspected of having symptoms associated with MASP-2-dependent complement activation with a therapeutically effective amount sufficient to reduce or at least partially reduce the symptoms of the disease. In both prophylactic and therapeutic regimens, a composition comprising a MASP-2 inhibitor can be administered in a dosage amount until satisfactory therapeutic results are achieved in the subject. MASP-2 Inhibition The application of the compositions of the present invention is accomplished by a single administration of the compositions, or a limited order of administration, for the treatment of acute conditions such as reperfusion injury or other traumatic injuries. Alternatively, the composition may be administered at intervals of time for the treatment of chronic conditions, such as arthritis or psoriasis.

The compositions and methods of the present invention can be used for inflammation and osteoporosis by diagnostic and therapeutic medical and surgical procedures in general. To inhibit this progression, the MASP-2 inhibiting composition of the present invention can be used before and after surgery. As used herein, "before and after surgery" means that the patient is treated before and / or during and / or after surgery, i.e., before, during and after surgery, before and after surgery, Intraperitoneal, intraperitoneal, intraperitoneal, intraperitoneal, intraperitoneal, intraperitoneal, intraperitoneal, intraperitoneal, intrathecal, The pre-operative application may be performed by local administration of the composition to the surgical or surgical site, such as injection of the site or continuous or intermittent perfusion or systemic administration. Methods suitable for delivery of MASP-2 inhibitor solutions before and after topical surgery are described in U.S. Pat. 6,420,432, Demopulos and 6,645,168, Demopulos. Suitable methods for local delivery of a cartilage protecting composition comprising a MASP-2 inhibitor (s) are disclosed in PCT patent application WO 01/07067 A2. Methods and compositions suitable for systemic delivery of a cartilage protecting agent comprising a MASP-2 inhibitor (s) are disclosed in PCT patent application WO 03/063799 A2.

VI. Example

The following examples are intended to illustrate the best mode contemplated for carrying out the invention and are not intended to limit the scope of the invention. All references cited herein are expressly incorporated by reference.

Example 1

This example demonstrates that MASP-2 (MASP-2 - / -) deficiency or MApl9 (MApl9 + / +) produces sufficient mouse attention.

Materials and methods : The targeting vector pKO-NTKV 1901 was designed to disrupt three exons encoding the C-terminus of murine MASP-2 containing an exon encoding the serine protease domain (FIG. 4). PKO-NTKV 1901 was used to transfect the murine ES cell line E14.1a (SV129 Ola). Neomycin-resistant and thymidine kinase-sensitive clones were selected. 600 ES clones were screened, where four different clones were identified and proved to contain the expected selective targeting and recombination by Southern blots (FIG. 4). Chimeras were generated from these four positive clones by embryonic transfer. The chimeras were backcrossed in the genetic background C57 / BL6 to produce transgenic males. Genetically-inserted males were crossed with females to produce F1, which represents the heterozygosity of the MASP-2 gene, in which 50% of the offspring were destroyed. The heterozygous MASP-2 deficient progeny were produced by heterologous crossing of the heterozygous mice, resulting in a 1: 2: 1 ratio of heterozygous and wild-type mice, respectively.

Results and phenotypes : Results Allelic homozygous MASP-2 - / - deficient mice were found to be viable and fertile, and Southern blot to confirm correct targeting, Northern blot to confirm absence of MASP-2 mRNA, and MASP- 2 &lt; / RTI &gt; protein (data not shown) by Western blot to confirm the absence of the MASP-2 protein. The presence of MApl9 mRNA and absence of MASP-2 mRNA was further confirmed using time-lysis RT-PCR on a LightCycler instrument. MASP-2 - / - mice did not continue to express MAp19, MASP-1, and MASP-3 mRNA and protein as expected (data not shown). The presence and absence of mRNAs in the MASP-2 - / - mice for the promoter, factor B, factor D, C4, C2, and C3 was assessed by LightCycler analysis and was consistent with that of the wild-type progeny control (data not shown) . Plasma of homozygous MASP-2 - / - mice was totally deficient in lectin-pathway-mediated complement activation and alternative pathway complement activation (Example 2).

Generation of MASP-2 - / - note on pure C57BL6 background: MASP-2 - / - mice were backcrossed with pure C57BL6 strain to generate nine mice before using MASP-2 - / - strain as experimental animal model.

Example 2

This example demonstrates that MASP-2 is required for complement activation through complement and lectin pathways.

Methods and Materials :

Lectin pathway-specific C4 cleavage assays : C4 cleavage assays bind to L-picoline. A method for measuring the lectin pathway activation result from lipidic acid (LTA) of S. aureus is described in Petersen, et al., J. Immunol. Methods 257: 101 (2001). The assay (Example 11) was adapted to measure lectin pathway activation by MBL by plating plates of LPS and mannan or zymosan before adding MASP-2 - / - mouse serum as described below. The method was modified to eliminate the possibility of C4 splitting due to the classical pathway. This was achieved using a sample dilution buffer containing 1 M NaCl, allowing lectin pathway recognition components to bind to their ligands with high affinity but preventing intrinsic C4 activation, thus contributing to the involvement of the classical pathway by degradation of the C1 complex . In summary, serum samples (diluted with high salt (1 M NaCl) buffer) are added to the ligand-coated plates in the modified assay, and then a certain amount of purified C4 is added in buffer at physiological salinity. The bound recognition complex containing MASP-2 degrades C4 and attaches C4b.

Method of measurement:

1) Nunc Maxisorb microliter plates (Maxisorb, Nunc, Cat. No. 442404, Fisher Scientific) the 1㎍ / ㎖ met (M7504 Sigma) or coating buffer _{(15 mM Na 2 CO 3,} 35 mM NaHCO 3, pH 9.6) (E. G., Those listed below) diluted with &lt; / RTI &gt;

The following reagents were used for the assay:

a. (M7504 Sigma 100 [mu] l coated buffer solution at 1 [mu] / well):

b. Zymosan (1 mu g / well zidovudine (Sigma) 100 mu l coated buffer solution);

c. LTA (1 占 퐂 / well of 100 占 퐇-coated buffer solution or 2 占 퐂 / well of 20 占 퐇 of methanol solution)

d. 1 [mu] g of H-picoline-specific Mab 4H5-coated buffer solution

e. PSA (2 쨉 g / well 100 袖 l coating buffer solution) of Aerococcus viridans

f. 100 [mu] l / well of formalin-fixed S. A coating buffer solution of S. aureus DSM20233 (OD ₅₅₀ = 0.5).

2) Plates were incubated overnight at 4 ° C.

3) After overnight incubation, the remaining protein binding sites were incubated with 0.1 mM HSA-TBS blocking buffer (0.1% (w / v) HSA in 10 mM Tris-CL solution, 140 mM NaCl, 1.5 mM NaN ₃ , pH 7.4), and the plates were then washed three times with TBS / tween / Ca ²⁺ (TBS, 0.05% Tween 20 and 5 mM CaCl ₂ , 1 mM MgCl ₂ , pH 7.4) .

4) The sera samples tested were diluted with MBL-binding buffer (1 M NaCl), diluted samples were added to the plates and incubated overnight at 4 ° C. The wells containing the buffer solution were used only as a negative control.

5) After overnight incubation at 4 ° C, the plate was washed three times with TBS / tween / Ca ²⁺ . Human C4 (1 ug / ml BBS diluted solution (4 mM barbitol, 145 mM NaCl, 2 mM CaCl ₂ , 1 mM MgCl ₂ , pH 7.4) at 100 ㎕ / well was added to the plate and cultured at 37 캜 for 90 minutes Respectively. Plates were washed again with TBS / tween / Ca ²⁺ .

6) C4b attachment was detected with alkaline phosphatase-conjugated chicken anti-human C4c (1: 1000 diluted TBS / tween / Ca ²⁺ solution), which was added to the plate and incubated at room temperature for 90 minutes. Plates were washed three more times with TBS / tween / Ca ²⁺ .

7) Alkaline phosphatase was added to 100 μl of ρ-nitrophenyl phosphate substrate solution, incubated at room temperature for 20 minutes, and then detected by reading OD ₄₅₀ in a microliter plate reader.

Results : FIGS. 6A-B show the results of a comparison between MASP-2 +/- (confluent) and MASP-2 - (closed triangles) (Fig. 6B). FIG. 6C shows the effect of C4b adherence on MASP-2 - / + mice (n = 5) and MASP-2 - / - mice (n = 4) compared to wild type mice Exhibit relative C4 converting enzyme activity on plates coated with zymosan (white bars) or mannan (shaded bars). The error bar represents the standard deviation. As shown in Figures 6A-C, the plasma of MASP-2 - / - mice is a complete deficiency in lectin-pathway mediated complement activation on mannan and zymosan coated plates. These results clearly demonstrate that MASP-2, rather than MASP- or MASP-3, is the effector component of the lectin pathway.

Measurement of C3b adhesion :

1) Nunc Maxisorb microliter plates (Maxisorb, Nunc, Cat. No. 442404, Fisher Scientific) the 1㎍ / ㎖ met (M7504 Sigma) or coating buffer _{(15 mM Na 2 CO 3,} 35 mM NaHCO 3, pH 9.6) (E. G., Those listed below) diluted with &lt; / RTI &gt;

2) The residual protein binding site was incubated with 0.1% HSA-TBS blocking buffer (10 mM Tris-CL solution, 0.1 mM (w / v) HSA, 140 mM NaCl, 1.5 mM NaN ₃ , pH 7.4) Saturated &lt; / RTI &gt; plate.

3) Plates were washed with TBS / tw / Ca ⁺⁺ (TBS, 0.05% Tween 20 and 5 mM CaCl ₂ ) and diluted BBS was incubated with serum samples (4 mM barbitol, 145 mM NaCl, 2 mM CaCl ₂ , mM MgCl _2, was added to pH 7.4). The wells containing the buffer solution were used as a negative control. Serum samples from the control set obtained from wild-type or MASP-2 - / - mice were deficient in C1q before being used for measurement. C1q-deficient mouse sera are prepared using protein-A-coupled dynabeads (Dynal Biotech, Oslo, Norway) coated with levit anti-human C1q IgG (Dako, Glostrup, Denmark) according to the manufacturer's instructions.

4) After overnight incubation at 4 ° C, the cells were washed with TBS / tw / Ca ⁺⁺ and the transformed and bound C3 was incubated with polyclonal anti-human-C3c antibody (TBS / tw / Ca ⁺⁺ diluted 1: 1000 Dako A 062). The secondary antibody is a goat anti-rabbit IgG (whole molecule) conjugated to alkaline-phosphatase (Sigma Immunochemicals A-3812) diluted 1: 10,000 in TBS / tw / Ca ⁺⁺ . The entire alternative complement pathway (AP) is determined by the addition of 100 [mu] l substrate solution (Sigma Fast p-nitrophenylphosphate tablet set, Sigma) and incubation at room temperature. Hydrolysis was monitored quantitatively by measuring absorbance at 450 nm in a microliter plate reader. Standard curves were generated for each analysis using serial dilution plasma / serum samples.

Results : The results shown in Figs. 7A and 7B were from molds forming a pool of several mice. The open triangle represents the MASP-2 + / + serum, the black circle represents the Cq-deficient MASP-2 + / + serum and the open square represents the MASP-2 - / - Lt; / RTI &gt; As shown in FIG. 7A-B, sera from MASP-2 - / - mice tested with the C3b attachment assay show low levels of C3 activation on mannan (FIG. 7A) and zymosan (FIG. 7B) coated plates. This result clearly demonstrates that MASP-2 needs to contribute to the initial C3b generation in C3 to initiate an alternative complement pathway. This is a surprising result from the general point of view that complement Factor C3, Factor B, Factor D, and Proprin form an independent functional alternative pathway, C3 is an abundance of C3b molecules that produce fluid phase transition enzyme iC3Bb and attachment C3b molecules on the activated surface, And undergo spontaneous conformational changes in the "C3b-like" form.

Recombinant MASP-2 reconstitutes lectin pathway-dependent C4 activation in MASP-2 - / - mouse serum

In order to establish that the absence of MASP-2 is a direct cause of lectin pathway-dependent C4 activation loss in MASP-2 - / - mice, the effect of addition of recombinant MASP-2 protein on serum samples was assessed by C4 split assay . The functionally active murine MASP-2 and the catalytically inactive murine MASP-2A (the active-site serine residue in the serine protease domain replaced the alanine residue) produced recombinant proteins and were purified as in Example 5. 4 MASP-2 - / - mice were preincubated with increasing protein concentrations of recombinant murine MASP-2 or inactive recombinant murine MASP-2A, and the C4 converting enzyme activity was measured as described above.

Results : As shown in Figure 8, functionally active murine recombinant MASP-2 protein (open triangles) was added to the serum of MASP-2 - / - mice to induce lectin pathway-dependent C4 activation in a protein concentration- Where the catalytically inactive murine MASP-2A protein (marked) failed to restore C4 activation. The results of Figure 8 were normalized for C4 activation observed with wild-type mouse serum (dotted line) that formed the pool.

Example 3

This example demonstrates the generation of murine inset mice expressing murine MASP-2 - / -, MApl9 + / +, or human MASP-2 insertional genes (murine MASP-2 knock-out and human MASP-2 knock- ).

Material and methods: a mini gene (a so-called "mini hMASP-2") coding for the human MASP-2 (SEQ ID NO: 49, Figure 5) is built to contain the promoter region of the human MASP 2 gene, the following 8 exons of the (Exons 1 to 3) followed by a cDNA sequence representing the coding sequence for the full-length MASP-2 protein, thereby encoding the full-length MASP-2 protein induced by the endogenous promoter. The mini hMASP-2 construct is injected into the fertilized egg of MASP-2 - / -, which expresses human MASP-2 by gene insertion and replaces deficient murine MASP2.

Example 4

This example illustrates the separation of human MASP-2 protein within the enzyme precursor form of human serum.

Methods of Human MASP-2 Isolation : Human serum MASP-2 isolation methods are described in Matsushita et al., J. Immunol. 165: 2637-2642, 2000. Briefly, human serum was incubated in a yeast mannan-Sepharose column using 10 mM imidazole buffer (pH 6.0) containing 0.2 M NaCl, 20 mM CaCl ₂ , 0.2 mM NPGB, 20 μM ρ-APMSF, and 2% mannitol I passed it. The MASP-1 and MASP-2 enzyme precursor complexes with MBL were eluted with the buffer containing 0.3 M mannose. To separate the enzyme precursors MASP-1 and MASP-2 from MBL, the product containing the complex was applied to anti-MBL-Sepharose, and then the MASP was incubated with imidazole buffer containing 20 mM EDTA and 1 M NaCl Respectively. Finally, the enzyme precursors MASP-1 and MASP-2 were separated from each other by passing through anti-MASP-1-Sepharose in the same buffer used as anti-MBL-Sepharose. MASP-2 was recovered from the eluate but MASP-1 was eluted with 0.1 M glycine buffer (pH 2.2).

Example 5

This example illustrates the recombinant expression and protein production of the catalytically activated mutant forms of the polypeptide-inducible recombinant full-length human, retroviral, and murine MASP-2, MASP-2, and MASP-2.

Expression of full-length human, murine and ret-MASP-2 :

The full-length cDNA sequence of human MASP-2 (SEQ ID NO: 4) was subcloned into the mammalian expression vector pCI-Neo (Promega), which induces eukaryotic expression under control of the CMV enhancer / promoter region (Kaufman RJ et al., Nucleic Acids Research 79: 4485-90, 1991; Kaufman, Methods in Enzymology, 185: 531-66 (1991)). The full length mouse cDNA (SEQ ID NO: 50) and Rett MASP-2 cDNA (SEQ ID NO: 53) were subcloned into the pED expression vector, respectively. The MASP-2 expression vector was transfected into the cohesive Chinese hamster ovary cell line DXB1 using a standard calcium phosphate transfection procedure (Maniatis et al., 1989). Transfected cells with these constructs grow very slowly and imply that the encoded protease is cytotoxic.

In another approach, the minigene construct (SEQ ID NO: 49) containing the human cDNA of MASP-2 was transiently transduced into Chinese hamster ovary cells (CHO) by its endogenous promoter. The human MASP-2 protein was secreted into the culture medium and isolated as follows.

Expression of full-length catalytic inactive MASP-2 :

Rationale: MASP-2 is activated by autocatalytic cleavage after the recognition sub-component MBL or picoline (L-picoline, H-picoline or M-picoline) binds to their individual carbohydrate patterns. Activation of MASP-2 by autocatalytic cleavage often occurs during the MASP-2 separation procedure from serum or purification procedure after recombinant expression. In order to obtain a more stable protein preparation to be used as an antigen, the catalytic inactive form of MASP-2 designed as MASP-2A is: Ret (SEQ ID NO: 55 Ser 617 with Ala 617); Mouse (SEQ ID NO: 52 Ser617 with Ala617); Or by replacing the serine residue present in the catalytic triad of the protease domain with the alanine residue in human (SEQ ID NO: 3 Ser618 with Ala618).

To generate catalytically inactive human and murine MASP-2A proteins, site-directed mutagenesis is carried out using oligonucleotides (Table 5). The oligonucleotides in Table 5 were designed to resolve regions of human and murine cDNA encoding enzymatically active serines, and oligonucleotides contain mismatches such that the serine codon changes to an alanine codon. For example, PCR oligonucleotides SEQ ID NO: 56-59 may be used in combination with human MASP-2 cDNA (SEQ ID NO: 4) to amplify the region from the initiation codon to the enzyme-active serine and serine to the termination codon, This leads to a fully open reading type of mutant MASP-2A containing the mutation to be Ala618. The PCR products were purified using agarose gel electrophoresis and band preparation and single adenosine overlap generation using a standard tailing procedure. Adenosine tail MASP-2A is followed by. And cloned into a pGEM-T ectopic vector transformed into E. coli.

The catalytic inactivation MASP-2A protein is produced by degrading and annealing these two oligonucleotides SEQ ID NO: 64 and SEQ ID NO: 65 for two minutes at an equimolar amount, heating to 100 DEG C and slow cooling to room temperature . The resulting disrupted fragment retains the Pst1 and Xba1 conforming ends and is inserted at the position of the Pst1-Xba1 fragment of the wild-type MASP-2 cDNA (SEQ ID NO: 53) to produce ret MASP-2A.

5 'GAGGTGACGCAGGAGGGGCATTAGTGTTT 3' (SEQ ID NO: 64)

5 'CTAGAAACACTAATGCCCCTCCTGCGTCACCTCTGCA 3' (SEQ ID NO: 65)

Human, murine and retest MASP-2A are further subcloned into the mammalian expression vector pED or pCI-Neo, respectively, or transferred to the Chinese hamster ovary cell line DXB1. As another approach, MASP-2 in the catalytic inactive form has been reported in Chen et al., J. Biol. Chem., 27 <5 (28): 25894-25902, 2001. In summary, a plasmid containing the full length human MASP-2 cDNA (Thiel et al., Nature 386: 506, 1997) is degraded by Xho1 and EcoR1 and the MASP-2 cDNA (SEQ ID NO: And cloned into the corresponding restriction sites of the viral transfer vector (Life Technologies, NY). The MASP-2 serine protease activation site at Ser618 is converted to Ala618 by substituting the native region amino acids 610-625 for the double-stranded oligonucleotides encoding the peptide region amino acids 610-625 (SEQ ID NO: 13) to generate an inactive protease domain To produce a full-length MASP-2 polypeptide. The structure of the expression plasmid containing the polypeptide region was derived from human Masp-2.

The following constructs are prepared using MASP-2 single peptide (residues 1-15, SEQ ID NO: 5) to secrete various domains of MASP-2. The construct expressing the human MASP-2 CUBI domain (SEQ ID NO: 8) is produced by PCR amplifying residues 1-121 coding region of MASP-2 (SEQ ID NO: 6) Response). The construct expressing the human MASP-2 CUBIEGF domain (SEQ ID NO: 9) is produced by PCR amplifying the MASP-2 (SEQ ID NO: 6) residue 1-166 coding region (corresponding to the N-terminal CUBIEGF domain ). The construct expressing the human MASP-2 CUBIEGFCUBII domain (SEQ ID NO: 10) is produced by PCR amplifying the MASP-2 (SEQ ID NO: 6) residue 1-293 coding region (corresponding to the N-terminal CUBIEGFCUBII domain ). The above-mentioned domains are amplified by PCR using Vent _R polymerase according to the established PCR method and pBS-MASP-2 as a template. The sense primer 5 'primer sequence (5'-CG GGATCC ATGAGGCTGCTGACCCTC-S ' SEQ ID NO: 34) introduces a BamHI restriction site (underlined) at the 5 'end of the PCR product. Antisense primers for each MASP-2 domain (Table 5) are designed to be introduced into the termination codon (bold) followed by an EcoRI site (underlined) at the end of each PCR product. When amplified, the DNA fragment is digested with BamHI and EcoRI and cloned into the corresponding region of the pFastBacl vector. The resulting structure is characterized by restriction mapping and is confirmed by dsDNA sequencing.

Table 5: MASP-2 PCR primers MASP-2 domain 5 'PCR primer 3 'PCR primer SEQ ID NO: 8 CUBI (aa SEQ ID NO: 6 of 1-121) 5 'CGGGATCCATGA GGCTGCTGACCCTC-3' (SEQ ID NO: 34) 5 'GGAATTCCTAGGCTGCATA3' (SEQ ID NO: 35) SEQ ID NO: 9 CUBIEGF (aa 1-166, SEQ ID NO: 6) 5 'CGGGATCCATGA GGCTGCTGACCCTC-3' (SEQ ID NO: 34) 5 'GGAATTCCTACAGGGCGC3' (SEQ ID NO: 36) SEQ ID NO: 10 CUBIEGFCUBII (SEQ ID NO: 6 of aa 1-293) 5 'CGGGATCCATGA GGCTGCTGACCCTC-3' (SEQ ID NO: 34) 5 'GGAATTCCTAGTAGTGGAT3' (SEQ ID NO: 37) SEQ ID NO: 4 human MASP-2 5 'ATGAGGCTGCTG ACCCTCCTGGGCC-3' (SEQ ID NO: 56) hMASP-2_ 5 'TTAAAATCACTAATTATG TTCTCGATC 3' (SEQ ID NO: 59) hMASP-2_ reverse SEQ ID NO: 4 human MASP-2 cDNA 5'CAGAGGTGACGC AGGAGGGGCAC3 '(SEQ ID NO: 58) hMASP-2_ala_ 5'GTGCCCCTCCTGCGTCAC CTCTG3 '(SEQ ID NO: 57) hMASP-2_alA reverse SEQ ID NO: 50 murine MASP-2 cDNA 5 'ATGAGGCTACTC ATCTTCCTGG3' (SEQ ID NO: 60) mMASP-2_ Trial 5 ' TTAGAAATTACTTATTAT GTTCTCAATCC3 ' (SEQ ID NO: 63) mMASP-2 & SEQ ID NO: 50 murine MASP-2 cDNA 5 'CCCCCCCTGCGT CACCTCTGCAG3' (SEQ ID NO: 62) mMASP-2_ala_ 5'CTGCAGAGGTGACGCAG GGGGGG3 '(SEQ ID NO: 61) mMASP-2_ala reverse

Recombinant eukaryotic expression of MASP-2 and protein production of enzyme-inactive mouse, rat, and human MASP-2A .

The MASP-2 and MASP-2A expression constructs are transfected into DXB1 cells using standard calcium phosphate transfection procedures (Maniatis et al., 1989). MASP-2A was produced in a serum-free medium and prevents products from being contaminated by other serum proteins. The medium was harvested every other day from the fusogenic cells (total 4 times). Each of the three recombinant MASP-2A levels was approximately 1.5 mg / l in culture medium.

MASP-2A Protein Purification : MASP-2A (the Ser-Ala mutation) is purified by affinity chromatography on MBP-A-agarose columns. This strategy enables rapid refinement without using additional tags. MASP-2A (Eastern fatigue dilution of 100-200 ml of medium, loading buffer (containing 50 mM Tris-Cl, pH 7.5, 150 mM NaCl and 25 mM CaCl ₂ ) was preincubated with 10 ml of loading buffer prior to MBP- Agarose affinity column (4 ml). It was then washed with 10 ml of additional loading buffer and the protein was eluted in 1 ml fractions (containing 50 mM Tris-Cl, pH 7.5, 1.25 M NaCl and 10 mM EDTA). The fraction containing MASP-2A was confirmed by SDS-polyacrylamide gel electrophoresis. If necessary, MASP-2A was further purified by ion-exchange chromatography on a Mono Q column (HR 5/5). The protein was dialyzed against 50 mM Tris-Cl (pH 7.5, containing 50 mM NaCl) and was administered to the equilibrated column in the same buffer. After washing, the bound MASP-2A was eluted with at least 10 ml of 0.05-1 M NaCl.

RESULTS : The 0.25-0.5 mg output of MASP-2A protein was obtained in 200 ml medium. The molecular mass of 77.5 kDa as measured by MALDI-MS was larger than the calculated value of unmodified polypeptide (73.5 kDa) by glycosylation. The glycan attachment of each N-glycosylation site accounts for the observed mass. MASP-2A migrates as a single band on SDS-polyacrylamide gel, demonstrating that they are not proteolytically processed upon biosynthesis. The weight-average molecular weight measured by equilibrium ultracentrifugation corresponds to the calculated value of the homodimer of the glycosylated polypeptide.

Production of Recombinant Human MASP-2 Polypeptide

Methods for the production of recombinant MASP-2 and MASP2A derived from another polypeptide are described in Thielens, NM, et al., J. Immunol. 166: 5068-5077, 2001. To summarize, Spodoptera frugiperda insect cells (Ready-Plaque Sf9 Cell, Novagen, Madison, Wis.) Were infected with Sf900II supplemented with 50 IU / ml penicillin and 50 mg / ml streptomycin Were grown and maintained in serum-free (Life Technologies) medium. Trichoplusia ni (High Five) insect cells (Jadwiga Chroboczek, Institut de Biologie Structurale, Grenoble, France) were cultured in TC100 medium supplemented with 50 IU / ml penicillin and 50 mg / ml streptomycin (Life Technologies, 10% FCS (Dominique Dutscher, Brumath, France)). Recombinant baculovirus was generated using the Bac-to-Bac system (Life Technologies). The bacmid DNA was purified using a Qiagen midiprep purification system (Qiagen) and used in Sf900II SFM medium (Life Technologies) for Sf9 insect cell transfer infection using cell pectin according to the manufacturer's instructions. The recombinant virus particles were collected after 4 days, and their potencies were measured by viral plaque assay. King and Possee, The Baculovirus Expression System: A Laboratory Guide, Chapman and Hall Ltd., London, pp. 111-114, 1992.

High-five cells (1.75 x 10 ⁷ cells / 175-cm ² tissue culture flask) were infected with recombinant virus containing MASP-2 polypeptide as infection multiplicity 2 in Sf900II SFM medium at 28 ° C for 96 hours. The supernatant was collected by centrifugation and diisopropylphosphorofluidate was added to give a final concentration of 1 mM.

The MASP-2 polypeptide is secreted into the culture medium. The culture supernatant was dialyzed against 50 mM NaCl, 1 mM CaCl ₂ , 50 mM triethanolamine hydrochloride, pH 8.1, and loaded on a Q-Sepharose high-speed flow column (Amersham Pharmacia Biotech) equilibrated in the same buffer at 1.5 ml / (2.8 x 12 cm). Elution is carried out by applying 1.2 liters of linear component to the same buffer solution of 35OmM NaCl. Fractions containing the recombinant MASP-2 polypeptide were identified by western blot analysis, adding (NH ₄ ) ₂ SO ₄ to 60% (w / v) and leaving overnight at 4 ° C. Pellets applied on the _{145 mM NaCl, 1 mM CaCl 2} , 50 mM triethanolamine hydrochloride, was resuspended in pH 7.4, the equilibrium of TSK G3000 SWG column in the same buffer (7.5 x 600 mm) (Tosohaas , Montgomeryville, PA) do. The purified polypeptide was concentrated (mw exclusion = 10,000) (Filtron, Karlstein, Germany) to 0.3 mg / ml using the supernatant method on a Microsep microcentrifuge.

Example 6

This example describes a method of producing polyclonal antibodies to MASP-2 polypeptides.

Materials and Methods :

MASP-2 Antigen : A polyclonal anti-human MASP-2 antiserum is produced by levitin immunized with the following isolated MASP-2 polypeptide: human MASP-2 isolated from serum (SEQ ID NO: 6) Example 4); MASP-2A (Example 4-5) containing recombinant human MASP-2 (SEQ ID NO: 6), inactive protease domain (SEQ ID NO: 13); And recombinant CUBI (SEQ ID NO: 8), CUBEGFI (SEQ ID NO: 9), and CUBEGFCUBII (SEQ ID NO: 10) (Example 5).

Polyclonal antibody : 6-week-old Levit, inoculated with BCG (bacillus Calmette-Guerin vaccine), was immunized by injection of 100 μg of MASP-2 polypeptide in 100 μg / ml sterile saline solution. Injections were performed every 4 weeks and antibody titers were monitored by ELISA assay (Example 7). The culture supernatant was collected by protein A affinity chromatography for antibody purification.

Example 7

This example illustrates a method for producing murine monoclonal antibodies against retroviral or human MASP-2 polypeptides.

Materials and Methods :

200 [mu] l of 100 [mu] g human or rat rMASP-2 or rMASP-2A polypeptide in complete Freund's adjuvant (Difco Laboratories, Detroit, Mich.) To 8-12 week old male A / J mice (Harlan, Houston, (PBS) solution (pH 7.4) was subcutaneously injected (same as in Example 4 or Example 5). At 2-week intervals, mice were subcutaneously injected with 50 [mu] g of human or retest rMASP-2 or rMASP-2A polypeptide in incomplete Freund's adjuvant. At week 4, mice were injected with a PBS solution of 50 [mu] g of human or rat rMASP-2 or rMASP-2A polypeptide and fused four days later.

For each fusion, a single cell suspension was prepared in the spleen of immunized mice and used for fusion with Sp2 / 0 multiple myeloma cells. 5 x 10 ⁸ Sp2 / 0 and 5 x 10 ⁸ spleen cells were grown in 50% polyethylene glycol (MW 1450) (Kodak, Rochester, NY) and 5% dimethylsulfoxide (Sigma Chemical Co., St. Louis, Mo.) &Lt; / RTI &gt; Cells were cultured in Iscove medium (Gibco, Grand Island, NY) supplemented with 10% bovine fetal serum, 100 units / ml penicillin, 100 ug / ml streptomycin, 0.1 mM hypoxanthine, 0.4 uM aminopterin and 16 uM thymidine ) &Lt; / RTI &gt; per suspension of 1.5 x 10 &lt; ⁵ &gt; 200 microliter of cell suspension was added to each well of about 20 96-well microflora plates. After about 10 days, the culture supernatant was collected for ELISA assay to determine the reactivity of the purified factor MASP-2.

ELISA Assay : Immulon 2 (Dynatech Laboratories, Chantilly, Va.) Wells of a microtitre plate were coated with 50 μl of purified hMASP-2 or 50 ng / ml rat rMASP-2 (or rMASP- . Low concentrations of MASP-2 for coating allow selection of high-affinity antibodies. After removing the coating solution with plate frying, 200 [mu] l of a PBS solution of BLOTTO (no-fat drying oil) was added to each well for 1 hour to block non-specific sites. One hour later, the wells were washed with buffer PBST (PBS, containing 0.05% Tween 20). 40 microliters of culture supernatant was collected from each fusion well and 50 microliters of BLOTTO was mixed and then added to each well of the microtitre plate. After 1 hour of incubation, the wells were washed with PBST. Bound murine antibodies were detected by reaction with red pepper peroxidase (HRP) conjugated goat anti-mouse IgG (Fc specific) (Jackson ImmunoResearch Laboratories, West Grove, Pa.) And diluted 1: 2,000 in BLOTTO . For color development, a peroxidase substrate solution containing 0.1% 3,3,5,5 tetramethylbenzidine (Sigma, St. Louis, Mo.) and 0.0003% hydrogen peroxide (Sigma) was added to each well for 30 minutes. The reaction was terminated by the addition of 50 [mu] l of 2M H ₂ SO ₄ / well. The optical density at 450 nm of the reaction mixture was read in a BioTek ELISA Reader (BioTek Instruments, Winooski, Vt.).

MASP-2 binding assay :

The culture supernatants tested positive in the MASP-2 ELISA assay can be tested by the binding assay to determine the MASP-2 inhibitor binding affinity for MASP-2. Similar assays can be used to determine whether inhibitors bind to other antigens in the complement system.

(PBS) of MASP-2 (20 ng / 100 μl / well, Advanced Research Technology, San Diego, Calif.) Was inoculated in a polystyrene microliter plate well (96-well plate binding plate, Corning Costa, 0.0 &gt; 4 C &lt; / RTI &gt; at pH 7.4. After aspiration of the MASP-2 solution, the wells were blocked with PBS containing 1% bovine serum albumin (BSA; Sigma Chemical) for 2 hours at room temperature. Wells without MASP-2 coating were used as background control. Aliquots of various concentrations of hybridoma supernatants in the blocking solution or purified anti-MAASP-2 MoAbs were added to the wells. After incubation for 2 hours at room temperature, the wells were extensively rinsed with PBS. MASP-2-conjugated anti-MASP-2 MoAb was detected by addition of blocking solution of peroxidase-conjugated goat anti-mouse IgG (Sigma Chemical) and incubated for 1 hour at room temperature. Plates were again thoroughly rinsed with PBS and 100 μl of 3,3 ', 5,5'-tetramethylbenzidine (TMB) substrate (Kirkegaard and Perry Laboratories, Gaithersburg, MD) was added. The reaction of TMB was stopped with 100 μl of 1M phosphoric acid, and the plate was read at 450 nm with a microplate reader (SPECTRA MAX 250, Molecular Devices, Sunnyvale, Calif.).

The ability to inhibit complement activation was tested for the culture supernatant of the positive well by a functional assay method such as C4 cleavage assay (Example 2). Cells in positive wells were cloned with limiting dilution. MoAbs was again tested for the reactivity of hMASP-2 in the ELISA assay described above. Selected hybridomas were grown in a spinner flask, and the culture supernatants used were collected for antibody purification by protein A affinity chromatography.

Example 8

This example illustrates the generation of human MASP-2 expressing MASP-2 - / - knockout mice for use as a screening model for MASP-2 inhibitors.

MATERIALS AND METHODS : MASP-2 - / - mice (Example 1) and MASP-2 - / - mice expressing human MASP-2 insert gene constructs (human MASP-2 knockin) And the offspring of murine MASP-2 - / -, murine MAp 19+, and human MASP-2 + were used to identify human MASP-2 inhibitors.

Such animal models can be used as test substrates for identification and efficacy of compositions containing MASP-2 inhibitors such as human anti-MASP-2 antibodies, MASP-2 inhibitory peptides and non-peptides, and MASP-2 inhibitors. For example, an animal model may be exposed to a compound or agent known to be capable of triggering MASP-2-dependent complement activation, and the MASP-2 inhibitor may be administered to the animal in a time and concentration sufficient to cause a reduction in the disease symptoms Is administered to an animal model.

In addition, murine MASP-2 - / -, MAp 19+, human MASP-2 + mice were used to generate cell lines containing one or more cell types involved in MASP- Can be used. The generation of continuous cell lines of the transgenic animals is well known in the art and is described, for example, in Mini, J.A., et al., Mol. Cell Biol, 5: 642-48, 1985.

Example 9

This example describes a method for producing human antibodies against human MASP-2 in a human MASP-2 and a MASP-2 knockout mouse expressing human immunoglobulin.

Materials and Methods :

MASP-2 - / - mice are generated as described in Example 1. Mice were constructed to express human MASP-2 as described in Example 3. Allogeneic splenic MASP-2 - / - mice and MASP-2 - / - mice expressing human MASP-2 were engineered to express at least a portion of the targeted destruction of the endogenous immunoglobulin heavy and light chain left and human immunoglobulin, respectively Were crossed with mice derived from embryonic stem cells. Preferably, a portion of the human immunoglobulin locus comprises unaligned sequences of heavy and light chain components. Both inactivation of the endogenous immunoglobulin gene and introduction of the exogenous immunoglobulin gene are accomplished by targeted homologous recombination. The gene insertion mammal resulting from this process can functionally rearrange the immunoglobulin component sequence and express a repertoire of antibodies of various isotype types encoded by the human immunoglobulin gene without expressing the endogenous immunoglobulin gene.

Production and characteristics of mammals with these properties are described, for example, in Thomson, AD, Nature 148: 1547-1553, 1994, and Sloane, B. F., Nature Biotechnology 14: 826, Mouse strains that have been genetically engineered such that the mouse antibody gene is inactivated and functionally replaced with a human antibody gene can be obtained (e.g., XenoMouse®, Abgenix, Fremont CA). The resultant offspring mice can produce human MoAbs against human MASP-2 suitable for use in human therapy.

Example 10

This example demonstrates the production and production of human adaptive murine anti-MASP-2 antibodies and antibody fragments.

Murine anti-MASP-2 monoclonal antibodies were generated in male A / J mice (Example 7). Murine antibodies reduce their immunogenicity by producing human chimeric IgG and Fab fragments of antibodies by replacing murine constant regions with human-adapted regions as described below, and in accordance with the present invention MASP-2- Lt; RTI ID = 0.0 &gt; complement-dependent &lt; / RTI &gt;

1. Cloning of anti-MAASP-2 variable region gene of murine hybridoma cells

Total RNA is isolated in hybridoma cell-secreted anti-MAASP-2 MoAb (Example 7) using RNAzol according to the manufacturer's protocol (Biotech, Houston, Tex.). A first strand cDNA is synthesized from total RNA using oligo dT as a primer. PCR was performed using an immunoglobulin constant C region-inducible 3 ' primer, denaturing the leader peptide of the murine VH or VK gene as a 5 ' primer or a set of primers derived from the first framework region. Anchored PCR is performed as described in the literature Chen and Platsucas (Chen, P. F., Scand. J. Immunol. 35: 539-549, 1992). For VK gene cloning, double stranded cDNA was prepared using Notl-MAKl primer (5'-TGCGGCCGCTGTAGGTGCTGTCTTT-3 'SEQ ID NO: 38). Cloned adapter ADl (5'-GGAATTCACTCGTTATTCTCGGA-3 'SEQ ID NO: 39) and AD2 (5'-TCCGAGAATAACGAGTG-3' SEQ ID NO: 40) are ligated to both 5 'and 3' ends of double stranded cDNA. The adapter at the 3 'end is removed by Not1 degradation. The degradation product is then used for PCR as a template with AD1 oligonucleotide as the 5 'primer and MAK2 (5'-CATTGAAAGCTTTGGGGTAGAAGTTGGTCGAGAGTTGGTC-3' SEQ ID NO: 41) as the 3 'primer. A DNA fragment of about 500 bp is cloned into pUC19. Several clones are selected for sequence analysis to demonstrate that the cloned sequence contains the expected murine immunoglobulin constant region. The Not1-MAK1 and MAK2 oligonucleotides were derived in the VK region and were 182 and 84 bp downstream from the original base pair of the C-kappa gene, respectively. The clones were selected including complete VK and leader peptide.

For cloning of the VH gene, the double-stranded cDNA was prepared using Notl-MAGl primer (5'-CGCGGCCGCAGCTGCTCAGAGTGTAGA-3 'SEQ ID NO: 42). The loosely coupled adapters AD1 and AD2 are ligated to both the 5 'and 3' ends of the double stranded cDNA. The adapter at the 3 'end is removed by Not1 degradation. The degradation product is used for PCR as a template with AD1 oligonucleotide and MAG2 (5'-CGGTAAGCTTCACTGGCTCAGGGAAATA-3 'SEQ ID NO: 43) as a primer. And cloned into DNA fragment pUC19 of 500 to 600 bp in length. The Not1-MAG1 and MAG2 oligonucleotides were derived from the murine C [gamma] .7.1 region and were 180 and 93 bp down from the initial bp of the murine C [gamma] .7.1 gene, respectively. The clones were selected to include the complete VH and leader peptide.

2. Construction of expression vectors for chimeric MASP-2 IgG and Fab.

The cloned VH and VK genes described above are used as templates in the PCR reaction and added to the 5 'end of the Kozak consensus sequence and the nucleotide sequence of the 3' end of the splice donor. After analysis to confirm the absence of PCR errors in the sequence, the VH and VK genes are inserted into expression vector cassettes containing human C. gamma 1 and C. kappa, respectively, to yield pSV2neoVH-huC gamma 1 and pSV2neoV-huC gamma. The CsCl component-purified plasmid DNA of the heavy- and light-chain vectors is used for the transfection of COS cells by electroporation. After 48 hours, the culture supernatant was tested by ELISA to confirm the presence of approximately 200 ng / ml chimeric IgG. Cells were harvested and total RNA was prepared. The first strand cDNA is synthesized from total RNA using oligo dT as a primer. This cDNA is used as a template in PCR to generate Fd and kappa DNA fragments. For the Fd gene, PCR was performed using 5'-AAGAAGCTTGCCGCCACCATGGATTGGCTGTGGAACT-3 '(SEQ ID NO: 44) and CHI-inducible 3' primer (5'-CGGGATCCTCAAACTTTCTTGTCCACCTTGG-3 'SEQ ID NO: 45) as 5' primers . The DNA sequence was confirmed to contain the complete VH and CH1 domains of human IgG1. After digestion with the appropriate enzymes, the Fd DNA fragment was inserted into HindIII and BamHI restriction sites of the expression vector cassette pSV2dhfr-TUS to yield pSV2dhfrFd. The pSV2 plasmid is available and consists of DNA fragments from a variety of sources: pBR322 DNA (thin line) contains a DNA replication (pBR ori) of pBR322 origin and a lactamase ampicillin resistance gene (Amp); SV40 DNA exhibited by the broad hatching and marking includes DNA replication of SV40 origin (SV40 ori), early promoters (5 'dhfr and neo genes), and polyadenylation signals (3' dhfr and neo genes). The SV40 -induced polyadenylation signal (pA) is located at the 3 ' end of the Fd gene.

For the kappa gene, PCR is performed using 5'-AAGAAAGCTTGCCGCCACCATGTTCTCACTAGCTCT-3 '(SEQ ID NO: 46) and CK-inducible 3' primer (51-CGGGATCCTTCTCCCTCTAACACTCT-31 SEQ ID NO: 47) as 5 'primers . The DNA sequence is found to contain complete VK and human CK regions. After digestion with the appropriate restriction enzymes, the kappa DNA fragment is inserted into the HindIII and BamHI restriction sites of the expression vector cassette pSV2neo-TUS to yield pSV2neoK. Expression of both the Fd and kappa genes is induced by the HCMV-inducible enhancer and promoter components. Because the Fd gene does not contain cysteine amino acid residues involved in the disulfide bond in the chain, the recombinant chimeric Fab includes non-covalently linked heavy- and light chains. This chimeric Fab was designed as a cFab.

To obtain a recombinant Fab with heavy and light chain disulfide bonds, the Fd gene was extended to include the coding sequence of an additional 9 amino acids (EPKSCDKTH SEQ ID NO: 48) from the hinge region of human IgG1. The BstEII-BamHI DNA portion encoding the 30 amino acid at the 3 'end of the Fd gene was replaced with the DNA portion encoding the extended Fd, resulting in pSV2dhfrFd / 9aa.

3. Expression and purification of chimeric anti-MASP-2 IgG

To generate chimeric anti-MASP-2 IgG secretory cell lines, NSO cells were infected with pSV2neoVH-huC.phi. And pSV2neoV-huC kappa of plasmid DNAs purified by electroporation. Transfer infected cells were selected in the presence of 0.7 mg / ml G418. Cells were grown in 250 ml Spinner flasks using serum-containing medium. The culture supernatant of 100 ml spinner culture was administered to a 10-ml PROSEP-A column (Bioprocessing, Inc., Princeton, N.J.). The column was washed with 10 bed volumes of PBS. The bound antibody was eluted with 50 mM citrate buffer, pH 3.0. The pH is adjusted to 7.0 by adding to the fraction containing the purified antibody of 1 M Hepes, pH 8.0, in the blood. Residual salts are removed by buffer exchange with PBS by Millipore membrane filtration (M.W. exclusion: 3,000). The protein concentration of the purified antibody is determined by the BCA method (Pierce).

4. Expression and purification of chimeric anti-MASP-2 Fab

For generation of cell lines that secrete chimeric anti-MASP-2 Fab, CHO cells were infected with pSV2dhfrFd (or pSV2dhfrFd / 9aa) and pSV2neo kappa of plasmid DNAs purified by electroporation. Transfected cells were selected for the presence of G418 and methotrexate. Selected cell lines amplified increased concentrations of methotrexate. Cells are single-cells subcloned by limiting dilution. High-production single-cell subcloned cell lines are then grown using a serum-free medium in a 100 ml spinner culture.

Chimeric anti-MAASP-2 Fab is purified by affinity chromatography using mouse anti-idiotype MoAb against MASP-2 MoAb. The anti-idiotypic MASP-2 MoAb is a specific MoAb binding that can be competed with human MASP-2 by immunizing mice with murine anti-MASP-2 MoAb conjugated with heat-labled hatchery hemorhinogen (KLH) Can be produced by screening. For purification, 100 ml of the supernatant of a spinner culture of CHO cells producing cFab or cFab / 9aa was added to an affinity column coupled with anti-idiotype MASP-2 MoAb. The column was thoroughly washed with PBS before the bound Fab was eluted with 50 mM diethylamine, pH 11.5. Residual salt was removed by the buffer exchange described above. The protein concentration of the purified Fab was determined by the BCA method (Pierce).

The ability of chimeric MASP-2 IgG, cFab, and cFAb / 9aa to inhibit the MASP-2-dependent complement pathway can be measured using inhibition assays (Example 2).

Example 11

This example demonstrates that in order to identify MASP-2 inhibitors capable of blocking MASP-2-dependent complement activation through L-picoline / P35, H-picoline, M- .

C4 cleavage assay : A C4 cleavage assay is described by Petersen, SV, et al., J. Immunol. Methods 257: 107, 2001, which measures the results of lectin pathway activation of lipidic tycoic acid (LTA) of S. aureus binding to L-picoline.

Reagents : Formalin-fixed S. aureous (DSM20233) is prepared as follows: Bacteria are grown overnight at 37 ° C in a tryptic soy blood medium, washed three times with PBS, and then incubated in PBS / 0.5% For 1 hour and further washed three times with PBS before being resuspended in coating buffer (15 mM Na ₂ CO ₃ , 35 mM NaHCO ₃ , pH 9.6).

Assay : Wells of a Nunc MaxiSorb microliter plate (Nalgene Nunc International, Rochester, NY) were coated with 100 μl of a coated buffer solution of formalin-fixed S. aureus DSM20233 (OD55Q = 0.5) and 1 ug of L- Solution. After overnight incubation the wells were blocked with a solution of 0.1% human serum albumin (HSA) in TBS (10 mM Tris-HCl, 140 mM NaCl, pH 7.4) and then incubated with 0.05% Tween 20 and 5 mM CaCl ₂ And washed with TBS (washing buffer). Human serum samples were diluted in 20 mM Tris-HCl, 1 M NaCl, 10 mM CaCl ₂ , 0.05% Triton X-100, 0.1% HSA, pH 7.4, which prevented the activation of endogenous C4, C1r, and C1s). Various concentrations of serum samples are added to MASP-2 inhibitors, including anti-MASP-2 MoAbs and inhibitory peptides. The diluted samples were added to the plates and incubated at 4 ° C overnight. After 24 hours, the plates were thoroughly washed with wash buffer and then 0.1 μg of purified human C4 (Dodds, AW, Methods Enzymol. 223: 46, 1993) are added to each well. To the wells are added 100 μl of 4 mM barbiturate, 145 mM NaCl, 2 mM CaCl ₂ , 1 mM MgCl ₂ , pH 7.4. After 1.5 h at 37 C, the plates were washed again and the C4b attachment was detected using an alkaline phosphatase-conjugated chicken anti-human C4c (Immunsystem, Uppsala, Sweden) and measured using the colorless substrate p-nitrophenyl phosphate do.

C4 Metabolism Measurements: The assay described above can be modified to measure lectin pathway activation through MBL by LSP and mannan-bearing coatings prior to addition to serum mixed with various MASP-2 inhibitors.

C4 measurement on H-picoline (Hakata Ag): The above-mentioned assay measures lectin pathway activation via H-picoline by coating with LSP and H-picoline prior to addition to serum mixed with various MASP-2 inhibitors Can be modified to measure.

Example 12

The following assays demonstrate the presence of classical pathway activation in wild-type and MASP-2 - / - mice.

Methods : The immune complexes were incubated for 1 hour at room temperature with 10 mM Tris, 140 mM NaCl, pH 7.4 solution of 0.1% human serum albumin in coated microliter plates (Maxisorb, Nunc, Cat. No. 442404, Fisher Scientific) And cultured overnight at 4 ° C in a Scottish Antibody Production Unit (Carluke, Scotland) diluted 1: 1000 with TBS / tween / Ca2 +. Serum samples are obtained in wild-type and MASP-2 - / - mice and added to coated plates. Control samples are prepared so that C1q is depleted in wild-type and MASP-2 - / - sera samples. C1q-deficient mouse sera are prepared using protein-A-coupled dynabeads (Dynal Biotech, Oslo, Norway) coated with levit anti-human C1q IgG (Dako, Glostrup, Denmark) according to the manufacturer's instructions. The plates were incubated at 37 DEG C for 90 minutes. The bound C3b is detected with a polyclonal anti-human-C3c antibody (Dako A 062) diluted 1: 1000 in TBS / tw / Ca ⁺⁺ . The secondary antibody is goat anti-rabbit IgG.

Results : Figure 9 shows the relative levels of C3b adhesion on plates coated with IgG in wild type serum, MASP-2 - / - serum, C1q-deficient wild type and C1q-deficient MASP-2 - / - sera. These results demonstrate that the classical pathway is intact in the MASP-2 - / - mouse strain.

Example 13

The following assays are used to test whether MASP-2 inhibitors block the classical pathway by analyzing the effects of MASP-2 inhibitors under the symptoms described by the classical pathway initiated by immune complexes.

METHODS : To test the effect of the MASP-2 inhibitor of the complement activation initiated by the classical pathway on immune complexes, a 50 μl sample containing 3 sets of 90% NHS was incubated with 10 μg / ml of the immunocomplex (IC) or PBS , And the same three samples (+/- IC) were included so as to contain 200 nM anti-proparD monoclonal antibody when cultured at 37 ° C. After two hours of incubation at 37 [deg.] C, 13 mM EDTA was added to all samples to terminate further complement activation, and the sample immediately cooled to 5 [deg.] C. The samples were then stored at -70 ° C before measuring the complement activation products (C3a and sC5b-9) using ELISA kits (Quidel, Catalog Nos. AO 15 and A009) according to the manufacturer's instructions.

Example 14

This example demonstrated that the lectin-dependent MASP-2 complement activation system is activated in the ischemia / reperfusion phase following abdominal aortic aneurysm repair.

Experimental basis and design : Ischemia-reperfusion injury was given to patients who underwent abdominal aortic aneurysm (AAA) repair, which was largely mediated by complement activation. We investigated the role of the lectin pathway of MASP-2-dependent complement activation in ischemia-reperfusion injury in patients with AAA repair. The mannan-binding lectin (MBL) in serum is used to measure the amount of MASP-2-dependent lectin pathway activation that occurs during reperfusion.

Patient Serum Sample Separation : A total of 23 patients with selective AAA restoration and eight control patients with major abdominal surgery were included in the study. For patients who underwent AAA restoration, systemic blood samples were collected from each patient's labor force (through the arterial line) at four fixed points during surgery: point 1: induction of anesthesia; Point 2: just before aortic clamping; Point 3: just before the aortic clamping removal; And time 4: at reperfusion. For control subjects with major abdominal surgery, systemic blood samples were taken at hypnotic induction and 2 hours after initiation of the procedure.

MBL level measurement : Each patient plasma sample was assayed for the level of mannan-binding lectin (MBL) using an ELISA technique.

Results : The results of this study are shown in FIG. 10, which shows the average percent change in MBL level (y axis) at various time points (x axis). The initial value of MBL is 100%, and thereafter decreases relatively. As shown in FIG. 10, AAA patients (n = 23) show a significant decrease in plasma MBL levels and an average of about 41% reduction at the time of ischemia / reperfusion after AAA. In contrast, we observed low MBL consumption in plasma samples in control subjects (n = 8) with major abdominal surgery.

The data provided provide a strong indication that the MASP-2-dependent lectin pathway of the complement system is activated on ischemia / reperfusion after AAA restoration. Decreased MBL levels seem to be related to ischemia-reperfusion injury, because the level of MBL is markedly and rapidly decreased when the cramped main vessel is reperfused after surgery. In contrast, the control serum of patients with major abdominal surgery without major ischemia-reperfusion injury shows only a slight decrease in MBL plasma levels. We conclude that the activation-dependent lectin pathway of MASP-2 on ischemic endothelial cells is a major pathologic factor of ischemia / reperfusion injury in terms of well-established contribution of complement activation in reperfusion injury. Thus, specific temporal blockade or reduction of the lectin pathway of MASP-2-dependent complement activation may be useful in the treatment of transient ischemic injuries, such as clinical surgery on myocardial infarction, intestinal infarction, burns, We expect to have a significant benefit in the effects of the effects.

Example 15

This example illustrates the use of the MASP-2 - / - strain as an animal model for testing MASP-2 inhibitors useful in the treatment of rheumatoid arthritis.

Background and Rationale : Murine arthritis model: K / BxN T cell receptor (TCR) gene insertion (tg) mouse is a recently developed inflammatory arthritis model (Kouskoff, V., et al., Cell 87: 811-822, 1996; Matsumoto, L, et al., Science 286: 1732-1735, 1999; Maccioni M. et al., J. Exp. Med. 195 (8) : 1071-1077, 2002). K / BxN mice develop their own autoimmune diseases with most of the clinical, histological and immunological characteristics of human RA (Ji, H., et al., Immunity 16: 157-168, 2002). Murine disorders are articular-specific, but are persistent by T after initiation, and B cells are autoreactive to glucose-6-phosphate isomerase ("GPI"), a ubiquitously expressed antigen. In addition, serum (or purified anti-GPI Igs) is transferred from arthritic K / BxN mice to healthy animals and causes arthritis within a few days. It has been shown that a pool of anti-GPI monoclonal antibodies to polyclonal anti-GPI antibodies or IgGl isotype induces arthritis when injected into healthy recipients (Maccioni et al., 2002). Because the murine model relates to human RA and the sera from patients with RA have been found to contain anti-GPI antibodies that are not found in normal individuals. C5-deficient mice have been tested in this system and have been shown to block arthritis development (Ji, H., et al., 2002, supra). There is strong inhibition of arthritis in C3 null mice, which is associated with an alternative pathway, but MBP-A null mice developed arthritis. In mice, however, the presence of MBP-C compensates for the loss of MBP-A.

Based on our observations that MASP-2 plays an essential role in the initiation of both lectins and alternative pathways, the K / BxN arthritic model is useful for screening effective MASP-2 inhibitors as RA therapeutics.

Methods : Serum of arthritic K / BxN mice was obtained at 60 days of age, pooled and injected (150-200 μl ip) into MASP-2 - / - recipient (Example 1); And control hatchlings were obtained at days 0 and 2 with or without MASP-2 inhibitors (MoAb, inhibitory peptides of the present invention, etc.). Normal mice were pre-treated with MASP-2 inhibitor 2 days before serum injection. Additional groups of mice were serially injected at day 0, and MASP-2 inhibitors were injected at day 6. Clinical indicators were given 1 point for each affected foot and 1/2 point for the slight swelling over time. The ankle thickness was measured with a caliper (the thickness was defined as the difference from the thickness at day 0).

Example 16

This example demonstrates an inhibition assay for complement-mediated tissue injury in an in vitro model of levitt's heart perfused with human plasma.

Background and Rationale : Activation of the complement system contributes to hyperacute rejection of xenografts. Previous studies have shown that hyperacute rejection can occur in the absence of anti-donor antibody through activation of alternative pathways (Johnston, PS, et al., Transplant Proc. 23: 877-879, 1991).

Methods : To determine if a detached anti-MAASP-2 inhibitor such as anti-MAsp-2 antibody (Example 7) could inhibit the complement pathway in tissue damage, anti-MAASP-2 MoAbs and antibody fragments were diluted Lt; / RTI &gt; in vitro model of isolated reptic heart perfused with human plasma. This model has previously been shown to induce damage to the levothyroxin by activating an alternative complement pathway (Gralinski, MR, et al., Immunopharmacology 34: 79-88, 1996).

Example 17

 This example illustrates a method of measuring neutrophil activation useful for determining an effective dose of a MASP-2 inhibitor for the treatment of symptoms associated with a lectin-dependent pathway in accordance with the methods of the present invention.

METHODS : Methods for the measurement of neutrophil elastase were described by Gupta-Bansal, R., et al., Molecular Immunol. 37: 191-201, 2000. In summary, the complex of elastase and serum α1-antitrypsin is measured by a two-site sandwich assay using antibodies against both elastase and α1-antitrypsin. Polystyrene microliter plates are coated at 4 [deg.] C with a PBS solution of anti-human elastasease antibody (binding site, Birmingham, UK) diluted 1: 500. After aspirating the antibody solution, the wells were blocked with PBS containing 0.4% HAS for 2 hours at room temperature. An aliquot (100 [mu] l) of the plasma sample treated or untreated with the MASP-2 inhibitor was added to the wells. After incubation for 2 hours at room temperature, the wells were thoroughly rinsed with PBS. The bound elastase-α1-antitrypsin complex is detected by the addition of a 1: 500 diluted peroxidase conjugate-α1-antitrypsin antibody blocking solution with 1 hour incubation at room temperature. After washing the plates with PBS, 100 [mu] l of TMB substrate aliquots were added. The reaction of TMB was stopped with the addition of 100 μl of phosphoric acid, and the plates were read at 450 nm in a microplate reader.

Example 18

This example illustrates an animal model for testing MASP-2 inhibitors useful for treating myocardial ischemia / reperfusion.

Methods : Myocardial ischemia-reperfusion models are described in Vakeva et al., Circulation 97: 2259-2267, 1998, and Jordan et al., Circulation 104 (12): 1413-1418, The model described was modified to be used in MASP-2 - / - and MASP-2 + / + mice as follows. Briefly, adult male mice were anesthetized. Cannulae were inserted into the carotid veins and organs, and respiration was maintained at 100% oxygen, adjusting to release between 3.5% and 5% CO ₂ with a rodent ventilator. Left thoracotomy was attempted and sutures were placed at 3 to 4 mm in origin of the left coronary artery. Animals were administered a MASP-2 inhibitor, such as an anti-MASP-2 antibody (e.g., between 0.01 and 10 mg / kg of the dose) 5 min before ischemia. Ischemia was initiated by tightening the suture surrounding the coronary artery, maintained for 30 minutes, and reperfused for 4 hours. The same Siamese-operated animals were prepared except for tightening the sutures.

Analysis of complement C3 attachment : After reperfusion, samples for immunohistochemistry were obtained from the central region of the left ventricle, fixed, and frozen at -80 DEG C until processing. Tissue sections were incubated with HRP-conjugated goat anti-retest C3 antibody. To identify MASP-2 inhibitors that reduce in vivo C3 adhesion, tissue sections were analyzed for the presence of C3 staining in the presence of anti-MASP-2 inhibitors compared to Siam-Surgery Control animals and MASP-2 - / - animals .

Example 19

This example demonstrates the use of the MASP-2 inhibitor as an animal model to test the ability of transplanted tissue to protect against ischemia / reperfusion injury.

Background / Rationale : Ischemia / reperfusion injury is known to occur within a donor organ during transplantation. The range of tissue damage, as demonstrated using various ischemic models and complement inhibitors such as soluble receptor type 1 (CR1), is related to the length of ischemia and is mediated by complement (Weisman et al., Science 249: 146-151 Pratt et al, Am. J. Path. 163 (4): 1457-1465, 2003). Animal transplantation models have been described by Pratt et al., Am. J. Path. 163 (4): 1457-1465, which discloses MASP-2 - / - mouse models that select the ability of MASP-2 inhibitors to protect from grafted tissue ischemia / reperfusion injury and / or MASP-2 + / + Modified for use as a model system. The perfusion of the donor kidney by the pre-implantation perfusion fluid provides an opportunity for introduction of the anti-MASP-2 inhibitor into the donor kidney.

Methods : MASP-2 - / - and / or MASP-2 + / + mice were anesthetized. The donor's left kidney was incised, ligated to the aorta in the head direction, and ligated into the kidney artery. (Portex Ltd, Hythe, UK) was inserted between the ligations and 5 ml of a solution of Soltron kidney perfusion solution (Baxter Health Care, UK) and MASP-2 inhibitor such as anti-MASP-2 monoclonal Antibody (range of doses .01 mg / kg to 10 mg / kg) was perfused for at least 5 minutes. Kidney transplants were performed and the progression of the mice was monitored.

Analysis of graft recipients: Kidney grafts were harvested at various time intervals, and tissue sections were analyzed using anti-C3 to determine the extent of C3 attachment.

Example 20

This example illustrates the use of a collagen-induced arthritis (CIA) animal model to test MASP-2 inhibitors useful for treating rheumatoid arthritis (RA).

Background and Rationale : Collagen-induced arthritis (CIA) is an autoimmune polyarthritis induced by rodent and primate strains susceptible to infection after immunization with native type II collagen and is recognized as a relevant model of human rheumatoid arthritis (RA) (Courtney et al., Nature 283: 666 (1980); Trenthan et al., J. Exp. Med., 146: 857 (1977)). Both RA and CIA are characterized by joint inflammation, panus formation, and cartilage and bone erosion. The CIA susceptible mutans DBA / 1LacJ was developed as a mouse CIA model that developed clinical vigorous arthritis after being immunized with bovine type II collagen (Wang et al., J. Immunol. 164: 4340-4347 (2000)). C5-deficient mouse strains were crossed with DBA / 1LacJ, and the results showed resistance to CIA arthritis development (Wang et al., 2000, supra).

Based on our observations that MASP-2 plays an essential role in the initiation of both lectins and alternative pathways, the CIA arthritic model is useful for screening MASP-2 inhibitors effective for use as RA therapeutics.

Method : MASP-2 - / - mice were generated as described in Example 1. MASP-2 - / - mice were crossed with mice derived from strain DBA / 1LacJ (The Jackson Laboratory). F1 and subsequent progeny were crossed to produce DBA / 1LacJ homozygous MASP-2 - / -.

Collagen immunization was performed as described in Wang et al., 2000, supra. Briefly, wild-type DBA / 1LacJ mice and MASP-2 - / - DBA / 1LacJ mice were treated with bovine type II collagen (BCII) or mouse type II collagen (MCII) (Elastin Products, Owensville, MO). Each mouse was injected with 200 ug CII and 100 ug mycobacteria into the dermis of the tail base. Mice were re-immunized 21 days later and the appearance of arthritis was examined daily. Arthritic indicators were evaluated over time for arthritis severity of each affected foot.

MASP-2 inhibitors may be administered at the time of collagen immunization, either systemically or locally to one or more joints, with an MASP-2 inhibitor such as anti-MASP-2 monoclonal antibody (dose range .01 mg / kg to 10 mg / kg) , And were selected in the wild type DBA / 1LacJ CIA mouse, and arthritic index was evaluated over time as described above. The anti-hMASP-2 monoclonal antibody as a therapeutic agent was simply assessed in the MASP-2 - / -, hMASP - + / + knock-in DBA / 1LacJ CIA mouse model.

Example 21

This example illustrates the use of the (NZB / W) F1 animal model to test MASP-2 inhibitors useful for treating immune-complex mediated glomerulonephritis.

Background and Rationale : New Zealand Black x New Zealand White (NZB / W) F1 mice develop self-immune syndrome with considerable similarity to human immunodeficiency-mediated glomerulonephritis. NZB / W F1 mice suffer glomerulonephritis without exception at 12 months of age. As described above, it has been demonstrated that complement activation plays a significant role in the pathogenesis of immune-complex mediated glomerulonephritis. This further demonstrated that administering anti-C5 MoAb to the NZB / W F1 mouse model significantly improved the course of glomerulonephritis (Wang et al., Proc. Natl. Acad Sci. 93: 8563-8568 )). Based on our observations that MASP-2 plays an essential role in the initiation of both lectins and alternative pathways, the NZB / W F1 animal model is useful for screening effective MASP-2 inhibitors for use as glomerular nephritis therapeutics.

Method : MASP-2 - / - mice were generated as described in Example 1. MASP-2 - / - mice were crossed with mice derived from NZB and NZW (The Jackson Laboratory), respectively. F1 and subsequent progeny to produce homozygous MASP-2 - / - in both NZB and NZW genetic backgrounds. To determine the role of MASP-2 in the pathogenesis of glomerulonephritis in this model, we examined the expression of MASP-2 in F1 subjects by crossing wild-type NZB x NZW mice or MASP-2 - / - NZB x MASP-2 - / - NZW mice The development of this disease was compared. Urinary samples were collected from MASP-2 + / + and MASP-2 - / - F1 mice at weekly intervals and urine protein levels were monitored for the presence of anti-DNA antibodies (Wang et al., 1996, supra). Histopathologic analysis of the kidney was performed to monitor interstitial matrix adhesion and glomerular nephritis development.

The NZB / W F1 animal model is useful for screening effective MASP-2 inhibitors for use as a glomerular nephritis treatment. At 18 weeks of age, an anti-MASP-2 inhibitor, such as an anti-MASP-2 monoclonal antibody (range of doses from .01 mg / kg to 10 mg / kg) is administered intraperitoneally at weekly or weekly intervals to wild type NZB / Respectively. The histopathological and biochemical markers of the-monitored glomerulonephritis are used to assess disease development in the mouse and to identify MASP-2 inhibitors useful for the treatment of this disease.

Example 22

This example illustrates the use of tubing loops as a model for testing MASP-2 inhibitors to prevent tissue damage results by extracorporeal circulation (ECC), such as cardiopulmonary bypass (CPB) circuitry.

Background and Rationale : As described above, patients with CPB-on-ECC may be exposed to blood by exposure to artificial surfaces of the cardiopulmonary circuit, but also by surface-independent factors such as surgical trauma and ischemia-reperfusion injury (Sir), S12-6, 1998. In the present study, we have investigated the effect of a systemic inflammatory response (Butler, J., et al., Ann. Thorac. Surg. 55: 552-9, 1993; Edmunds, LH, Ann. Thorac. ; Asimakopoulos, G., Perfurion 14: 269-77, 1999). It is further known that, due to the interaction between blood and the artificial surface of the CPB circuit, the alternative complement path plays a predominant role in complement activation in CPB circuits (Kirklin et al., 1983, 1986, Tactics). Thus, based on our observations that MASP-2 plays an essential role in the initiation of both lectin and alternative pathways, the tubing loop model is an effective MASP-2 inhibitor for use as a therapeutic agent for the prevention or treatment of cardiopulmonary- 2 &lt; / RTI &gt; inhibitor.

Methods : Gupta-Bansal et al., Molecular Immunol. 37: 191-201 (2000), a modification of the tubing loop model described above for cardiopulmonary bypass circuits has been used (Gong et al., J. Clinical Immunol. 16 (4): 222-229 1996). Briefly, blood is freshly collected in a healthy subject with 7 ml of gentle blood vessels (containing 7 units / ml of whole blood). A tube of polyethylene similar to that used in the CPB procedure (e.g., LD 2.92 mm; OD 3.73 mm, length 45 cm) was filled with 1 ml of blood and closed with a loop with small pieces of silicon tubing. The control tubing containing the hairpinized blood and 10 mM EDTA was included in the study as a background control. Samples and control tubing were rotated vertically in a bath at 37 占 폚 for 1 hour. After incubation, the blood sample was transferred to a 1.7 ml microfuse tube (containing EDTA) to a final concentration of 20 mM EDTA. The samples were centrifuged and the plasma was collected. MASP-2 inhibitors, such as anti-MASP-2 antibodies, were added to the hairpinized blood immediately before spinning. Plasma samples were added to the assay to determine the concentration of C3a and soluble C5b-9 (Gupta-Bansal et al., 2000, supra).

Example 23

This example demonstrates that rodent cecal ligation and tran- sition (CLP), which tests MASP-2 inhibitors useful for treating septic symptoms results, including the consequences of acute respiratory distress syndrome caused by septicemia or severe sepsis, septic shock, sepsis and systemic inflammatory response syndrome ) Model system.

Background and Rationale : As noted above, complement activation has been shown to play an important role in the pathogenesis of sepsis in a number of studies (Bone, RC, Annals. Internal. Med. 115: 457-469, 1991). The CLP rodent model is recognized as a model to simulate the clinical outcome of human sepsis and is considered a rational alternative model of human sepsis (Ward, P., Nature Review Immunology VoI 4: 133-142 (2004)). Recent studies have shown that anti-C5a antibody treatment of CLP animals reduces bacteremia and significantly improves survival (Huber-Lang et al., J. of Immunol. 169: 3223-3231 (2002)). Thus, based on our observations that MASP-2 plays an essential role in the initiation of both lectin and alternative pathways, the CLP rodent model is a MASP-2 inhibitor effective for use as a therapeutic agent for the prevention or treatment of sepsis or sepsis symptom outcome . &Lt; / RTI &gt;

Method : The CLP model was modified as follows from the model described in the document Huber-Lang et al., 2004, supra. MASP-2 - / - and MASP-2 + / + animals were anesthetized. A 2 cm median abdominal incision was made and the cecum was tightly ligated under the stromal valve to avoid bowel obstruction. The cecum was punctured with a 21-gauge needle. The abdominal incisions were sutured in layers with silk sutures and skin clips (Ethicon, Summerville, NJ). Immediately after CLP, animals were administered an injection of a MASP-2 inhibitor such as an anti-MASP-2 monoclonal antibody (dose range .01 mg / kg to 10 mg / kg). Anti-hMASP-2 monoclonal antibodies as therapeutic agents were readily evaluated in the MASP-2 - / -, hMASP - + / + knock-in CLP mouse model. Plasma of the mice was assayed for complement-induced anapylatoxin and levels of respiratory burst using the assay described in the document Huber-Lang et al., 2004, supra.

Example 24

This example demonstrates the identification of a high affinity anti-MASP-2 Fab2 antibody fragment that blocks MASP-2 activity.

Background and Rationale : MASP-2 is a fusion protein comprising: the binding site (s) of MBL and picoline, the serine protease catalytic site, the binding site of proteolytic substrate C2, the binding site of proteolytic substrate C4, the binding site of MASP- A MASP-2 cleavage site of activation, and a plurality of discrete functional domains comprising two Ca &lt; ⁺⁺ &gt; binding sites. Fab2 antibody fragments were identified to bind to MASP-2 with high affinity and the identified Fab2 fragments were tested for functional assays to determine if they blocked MASP-2 functional activity.

To block MASP-2 functional activity, the antibody or Fab2 antibody fragment must bind to or interfere with the structural epitope on MASP-2 required for MASP-2 functional activity. Thus, many or all of the high affinity binding anti-MASP-2 Fab2 can not inhibit MASP-2 functional activity unless they bind to a structural epitope on MASP-2 that is directly related to MASP-2 functional activity.

Functional assays measuring the inhibition of lectin pathway C3 converting enzyme formation are used to evaluate the "blocking activity" of anti-MASP-2 Fab2s. It is known that the primary physiological role of MASP-2 in the lectin pathway results in the production of the next functional component of the lectin-mediated complement pathway, the lectin pathway C3 converting enzyme. The lectin pathway C3 converting enzyme is a crucial enzymatic complex (C4bC2a) that proteolytically degrades C3 into C3a and C3b. MASP-2 is not a structural component of the lectin pathway C3 converting enzyme (C4bC2a); However, MASP-2 functional activity is required to produce two protein components (C4b, C2a) containing the lectin pathway C3 converting enzyme. In addition, all the distinct functional activities of the listed MASP-2 seem to be necessary for MASP-2 to produce the lectin pathway C3 converting enzyme. For this reason, a preferred assay for evaluating the "blocking activity" of anti-MASP-2 Fab2s is thought to be a functional assay measuring the inhibition of lectin pathway C3 converting enzyme formation.

Generation of high affinity Fab2s : A phage display library of human variable light and heavy chain antibody sequences and automated antibody selection techniques to identify Fab2s that react with the selected ligand of interest. High affinity for human MASP-2 protein (SEQ ID NO: 55) It is used to produce Fab2s. A known positive rat MASP-2 (~ 1 mg,> 85% pure) protein is used for antibody screening. Three rounds of amplification is used to select the antibody with the best affinity. About 250 different heat expressing antibody fragments are selected for ELISA screening. The high affinity hits are subsequently sequenced to determine the uniqueness of the other antibodies.

Forty unique anti-MASP-2 antibodies were purified and 250 μg of each purified Fab2 antibody to be used was used for characterization of MASP-2 binding affinity and complement pathway functional testing.

The assay used for the evaluation of inhibition (blocking) of anti-MASP-2 Fab2s activity

1. Assay for measuring inhibition of lectin pathway C3 converting enzyme formation:

Background: The lectin pathway C3 converting enzyme is an enzymatic complex (C4bC2a) that degrades C3 into two potential proinflammatory segments, anapylatoxin C3a and opsonin C3b. The formation of C3 converting enzyme appears to be a key step in the inflammatory mediating lectin pathway. MASP-2 is not a structural component of the lectin pathway C3 converting enzyme (C4bC2a); Thus, the anti-MAST-2 antibody (or Fab2) does not directly inhibit the activity of the predominant C3 converting enzyme. However, MASP-2 serine protease activity is required for the production of two protein components (C4b, C2a) containing the lectin pathway C3 converting enzyme. Thus, anti-MASP-2 Fab2, which inhibits MASP-2 functional activity (i. E., Anti-MAASP-2 Fab2 blockade) inhibits the new formation of lectin pathway C3 converting enzyme. C3 contains a specific and highly reactive thioester group as part of its structure. In the cleavage of the C3 by C3 converting enzyme in this assay, the thioester group on C3b can form a covalent bond with the hydroxyl or amino group on the molecule as a macromolecule fixed to the bottom of the plastic well through an ester or amide bond, Thereby facilitating the detection of C3b.

Yeast mannan is a known activator of the lectin pathway. In the following method to measure the formation of C3 converting enzyme, mannan coated plastic wells were incubated with diluted ret. Serum for 30 min at 37 [deg.] C to activate the lectin pathway. The wells were then washed and C3b immobilized on the wells was measured using a standard ELISA method. The amount of C3b produced in this assay directly reflects the new formation of the lectin pathway C3 converting enzyme. Anti-MASP-2 Fab2s at selected concentrations were tested in this assay for their ability to inhibit C3 conversion enzyme formation and resulting C3b production.

Way:

The 96-well Costa media binding plate was overnight cultured at 1 ug / 50 / / well at 5 째 C with mannan diluted in 50 mM carbonate buffer (pH 9.5). After overnight incubation, each well was washed three times with 200 [mu] l PBS. Wells were blocked with 100 [mu] l / well of PBS solution of 1% bovine serum albumin and incubated for 1 hour at room temperature with moderate mixing. Each well was washed three times with 200 [mu] l of PBS. Wherein -MASP-2 Fab2 samples of the selected concentration of Ca ⁺⁺ and Mg ⁺⁺ containing GVB buffer (4.O mM barbiturates, 141 mM NaCl, 1.O mM MgCl 2, 2.0 mM CaCl 2, 0.1% gelatin, pH 7.4). &Lt; / RTI &gt; 0.5% fetal calf serum was added to the sample at 5 ° C and 100 μl was transferred to each well. Plates were covered and cultured in a water bath at 37 ° C for 30 minutes to enable complement activation. The reaction was stopped by moving the plate in a 37 ° C water bath to an ice-water mixture vessel. Each well was washed five times with 200 [mu] l of PBS-Tween 20 (0.05% Tween 20 in PBS) and then twice with 200 [mu] l of PBS. 100 [mu] l / well of 1: 10,000 dilution primary antibody (levit anti-human C3c, DAKO A0062) was added to PBS containing 2.0 mg / ml bovine serum albumin and incubated for 1 hour at room temperature with moderate mixing. Each well was washed five times with 200 [mu] l PBS. 100 [mu] l / well of a 1: 10,000 dilution secondary antibody (peroxidase-conjugated goat anti-rabbit IgG, American Qualex A102PU) was added to PBS containing 2.0 mg / ml bovine serum albumin, And cultured at room temperature for 1 hour. Each well was washed five times with 200 [mu] l of PBS. 100 [mu] l / well peroxidase substrate TMB (Kirkegaard &amp; Perry Laboratories) was added and incubated at room temperature for 10 minutes. The peroxidase reaction was stopped by the addition of 100 μl / well of 1.0 MH ₃ PO ₄ and the OD ₄₅₀ was measured.

2. Measures to measure inhibition of MASP-2-dependent C4 cleavage

Background: The serine protease activity of MASP-2 is highly specific and only two protein substrates of MASP-2 have been identified; C2 and C4. C4 splitting produces C4a and C4b. Anti-MASP-2 Fab2 can bind to a structural epitope on MASP-2 directly related to C4 cleavage (e.g., MASP-2 binding site of C4; MASP-2 serine protease catalytic site) Inhibits cleavage functional activity.

Yeast mannan is a known activator of the lectin pathway. In the following method for measuring the C4 cleavage activity of MASP-2, mannan-coated plastic wells were incubated with diluted Rat serum for 30 min at 37 [deg.] C to activate the lectin pathway. The diluted fetal serum was supplemented with human C4 (1.0 [mu] g / ml), since only the primary antibody human C4 used in this ELISA assay was recognized. The wells were then washed and human C4b immobilized on a well using a standard ELISA method was immobilized. The amount of C4b produced in this assay is a measure of MASP-2 dependent C4 cleavage activity. Anti-MASP-2 Fab2 at selected concentrations was tested in this assay for their ability to inhibit C4 cleavage.

METHODS: 96-well Costa media binding plates were incubated overnight at 5 占 폚 with mannan (pH 9.5) diluted in 50 mM carbonate buffer at 1.0 占 퐂 / 50 占 퐇 / well. Each well was washed three times with 200 [mu] l PBS. Wells were blocked with 100 [mu] l / well of PBS solution of 1% bovine serum albumin and incubated for 1 hour at room temperature with moderate mixing. Each well was washed three times with 200 [mu] l of PBS. A sample of anti-MASP-2 Fab2 was incubated with GVB buffer (4.0 mM barbiturate, 141 mM NaCl, 1.0 mM MgCl ₂ , 2.0 mM CaCl ₂ , 0.1% gelatin, pH 7.4) containing Ca ⁺⁺ and Mg ⁺⁺ at a selected concentration of 5 Lt; / RTI &gt; 1.0 [mu] g / ml human C4 (Quidel) was added to these samples. 0.5% fetal calf serum was added to the sample at 5 째 C and 100 ㎕ was transferred to each well. Plates were covered and incubated at 37 ° C for 30 minutes to enable complement activation. The reaction was stopped by moving the plate in a 37 ° C water bath to an ice-water mixture vessel. Each well was washed five times with 200 [mu] l of PBS-Tween 20 (0.05% Tween 20 in PBS) and then twice with 200 [mu] l of PBS. 100 μl / well of 1: 700 diluted biotin-conjugated chicken anti-human C4c (Immunsystem AB, Uppsala, Sweden) was added to PBS containing 2.0 mg / ml bovine serum albumin (BSA) Time. Each well was washed five times with 200 [mu] l PBS. 100 [mu] l / well of 0.1 [mu] g / ml of peroxidase-conjugated streptavidin (Pierce Chemical # 21126) was added to PBS containing 2.0 mg / ml BSA and incubated for 1 hour at room temperature with gentle mixing in a shaker. Each well was washed five times with 200 [mu] l of PBS. 100 [mu] l / well peroxidase substrate TMB (Kirkegaard &amp; Perry Laboratories) was added and incubated at room temperature for 16 minutes. The peroxidase reaction was stopped by adding 100 μl / well of 1.0 MH ₃ PO ₄ and the OD ₄₅₀ was measured.

3. Measurement of binding of anti-retest MASP-2 Fab2 to 'natural' retest MASP-2

Background: MASP-2 is generally present in plasma as a MASP-2 dimer complex containing specific lectin molecules (mannose-binding protein (MBL) and picoline). Thus, when studying the binding of anti-MASP-2 Fab2 to MASP-2, which is a physiologic canal type, the binding assay in which Fab2 interacts with 'natural' MASP-2 in plasma rather than purified recombinant MASP- It is important to let them know. In this binding assay, the 'native' MASP-2-MBL complex of 10% fetal calf is first immobilized on the mannan-cloth well. Various anti-MASP-2 Fab2s binding affinities for immobilized 'natural' MASP-2 were studied using standard ELISA methods.

Methods: 96-well Costa high plate was incubated overnight at 5 째 C with 1 / / 50 / / well of mannan (pH 9.5) diluted in 50 mM carbonate buffer. Each well was washed three times with 200 [mu] l PBS. Wells were blocked with 100 μl / well of 0.5% non-fat dry oil PBST (PBS, 0.05% Tween 20) solution and incubated for 1 hour at room temperature with moderate mixing. Each well was washed three times with 200 μl of TBS / Tween / Ca ⁺⁺ wash buffer (Tris-buffered saline, 0.05% Tween 20, containing 5.0 mM CaCl ₂ , pH 7.4). (20 mM Tris, 1.0 M NaCl, 10 mM CaCl ₂ , 0.05% Triton-X100, 0.1% (w / v) bovine serum albumin, pH 7.4) in 10% fetal bovine serum was prepared in ice. 100 [mu] l / well were added and cultured overnight at 5 [deg.] C. Wells were washed three times with 200 [mu] l TBS / Tween / Ca ⁺⁺ wash buffer. The wells were then washed twice with 200 [mu] l PBS. Well of a selected concentration of 100 [mu] l / well diluted with GVB buffer (4.0 mM barbiturate, 141 mM NaCl, 1.0 mM MgCl ₂ , 2.0 mM CaCl ₂ , 0.1% gelatin, pH 7.4) containing Ca ⁺⁺ and Mg ⁺⁺ Anti-MASP-2 Fab2 was added and incubated for 1 hour at room temperature with moderate mixing. Each well was washed five times with 200 [mu] l PBS. PBS solution of 2.0 mg / ml bovine serum albumin in 100 μl / well of HRP-conjugated goat anti-Fab2 (Biogeneis Cat No. 0500-0099) diluted 1: 5000 was added and incubated for 1 hour at room temperature with moderate mixing. Each well was washed five times with 200 [mu] l PBS. 100 [mu] l / well peroxidase substrate TMB (Kirkegaard & Perry Laboratories) was added and incubated at room temperature for 70 min. The peroxidase reaction was stopped by the addition of 100 μl / well of 1.0 MH ₃ PO ₄ and the OD ₄₅₀ was measured.

result:

Approximately 250 different Fab2 reacting with the high affinity radiolabeled MASP-2 protein were selected by ELISA selection. These high affinity Fab2s were sequenced to determine the uniqueness of the other antibodies, and 50 unique anti-MASP-2 antibodies were purified and further analyzed. 250 &lt; RTI ID = 0.0 &gt; pg &lt; / RTI &gt; of each purified Fab2 antibody was used for MASP-2 binding affinity characterization and complement pathway functional testing. The results of this analysis are shown in Table 6.

Table 6: Anti-MASP-2 FAB2 blocking lectin pathway complement activation Fab2 antibody # C3 converting enzyme (IC50 (nM)) K _d C5 converting enzyme (IC50 (nM)) 88 0.32 4.1 ND 41 0.35 0.30 0.81 11 0.46 0.86 <2 nM 86 0.53 1.4 ND 81 0.54 2.0 ND 66 0.92 4.5 ND 57 0.95 3.6 <2 nM 40 1.1 7.2 0.68 58 1.3 2.6 ND 60 1.6 3.1 ND 52 1.6 5.8 <2 nM 63 2.0 6.6 ND 49 2.8 8.5 <2 nM 89 3.0 2.5 ND 71 3.0 10.5 ND 87 6.0 2.5 ND 67 10.0 7.7 ND

As shown in Table 6 above, 50 anti-MASP-2 Fab2s were tested and 17 Fab2s were tested for MASP-2 blockade that potentially inhibited the formation of C3 converting enzyme with an IC ₅₀ value equal to or less than 10 nM Fab2s Fab2 (34% positive hits). Identified 17 gaejung eight Fab2s that has an IC ₅₀ value in the range of nanomolar or less. Additionally, all 17 MASP-2 blocked Fab2s (Table 6) essentially show complete inhibition of C3 convertase formation in the lectin pathway C3 converting enzyme assay. Fig. 11A shows the result of C3 conversion-enzyme formation measurement of Fab2 antibody # 11, which is representative of other Fab2 antibodies tested, and the results are shown in Table 6. This is an important consideration because, in theory, the "blocking" Fab2 can partially inhibit MASP-2 function when each MASP-2 molecule is bound to Fab2.

Even if it is a known activator of the mannitol lectin pathway, theoretically, the presence of an anti-mannan antibody in the rat serum may enable to activate the classical pathway and produce C3b through the classical pathway C3 converting enzyme. However, the 17 respective blocking anti-MASP-2 Fab2s listed in this example potentially inhibited C3b production (> 95%), thus demonstrating the specificity of this assay for lectin pathway C3 converting enzyme.

Binding assays were performed with all 17 blocked Fab2s to calculate the respective apparent K _d . The results of the binding assay of anti-retest MASP-2 Fab2 and native retest MASP-2 against 6 blocking Fab2 are shown in Table 6. Figure 11B shows the results of the binding assay of Fab2 antibody # 11. Similar binding assays were performed with different Fab2, and the results are shown in Table 6. Generally, the apparent Kd obtained by the combination of each of the six Fab2 and 'native' MASP-2 is generally consistent with the IC50 value for Fab2 within the C3 converting enzyme functional assay. There is evidence that MASP-2 undergoes steric structural changes from 'inactive' to 'active' for activation of protease activity (Feinberg et al, EMBO J 22: 2348-59 (2003); Gal et al, J. Biol Chem. 280: 33435-44 (2005)). In the normal rat plasma used in the C3 conversion enzyme formation assay, MASP-2 appears primarily in the 'inert' enzyme source steric structure. In contrast, in the binding assay, MASP-2 exists as part of a complex with MBL bound to a fixed mannan; Thus, MASP-2 is in an 'active' conformation (Petersen et al., J. Immunol Methods 257: 107-16, 2001). As a result, it is not necessary to necessarily expect an exact match between the IC ₅₀ and the K _d for each of the 17 blocked Fab2 tested in these two functional assay methods. This is because Fab2 binds to different steric MASP-2 in each assay. Nevertheless, with the exception of Fab2 # 88, each of the other 16 Fab2 tested in both assays appears to have a fairly close match between the IC ₅₀ and the apparent K _d (Table 6).

Several blocked Fab2 were evaluated for MASP-2 mediated inhibition of C4. Figure 11C shows the results of a C4 cleavage assay, and represents the inhibition by Fab2 # 41, had IC ₅₀ = 0.81 nM (Table 6). As shown in Fig. 12, all of the Fab2 tested show inhibition of C4 cleavage with an IC ₅₀ similar to that obtained with C3 conversion assay (Table 6).

Even if it is a known activator of the manganese leptin pathway, theoretically, the presence of an anti-mannan antibody in the rat serum may enable to activate the classical pathway and produce C4b by the C1s-mediated cleavage of C4. However, several anti-MASP-2 Fab2 have been identified to potentially inhibit C4b production (> 95%), thus demonstrating the specificity of this assay for MASP-2 mediated C4 cleavage. C4 is specific to C3 as well as C3 and contains highly reactive thioester groups. In this assay, in the C4 cleavage by MASP-2, the thioester group on C4b may form a covalent bond with the hydroxyl or amino group on the molecule as a macro fixed to the bottom of the plastic well via an ester or amide bond, The detection of C4b is promoted. These studies clearly demonstrate the production of FAB2 in high-affinity to the ret MASP-2 protein, which functionally blocks both C4 and C3 converting enzyme activities, thus preventing lectin pathway activation.

Example 25

This example illustrates epitope mapping for several blocking anti-anti-MASP-2 Fab2 antibodies generated as described in Example 24.

Way:

As shown in Figure 13, all of the following proteins bearing the N-terminal 6X His tag were expressed in CHO cells using the pED4 vector:

Retinase MASP-2A, full-length MASP-2 protein (S613A), inactivated by a change in serine at the alanine active center;

Modified MASP-2K, full-length MASP-2 protein (R424K) modified to reduce self-activation;

An N-terminal fragment of CUBI-II, ret MASP-2, containing only CUBI, EGF-like and CUBII domains; And

N-terminal fragment of CUBI / EGF-like, retest MASP-2 containing only CUBI and EGF-like domains.

These proteins are purified from the culture supernatant by nickel-affinity chromatography as described above (Chen et al., J. Biol. Chem. 276: 25894-02 (2001)).

The C-terminal polypeptide (CCPII-SP) containing the serine protease domain of CCPII and ret MASP-2 was used as a thioredoxin fusion protein using pTrxFus (Invitrogen). Was expressed in E. coli. The protein was purified from the cell lysate using a thio-band affinity resin. The thioredoxin fusion partner was expressed in the empty pTrxFus as a negative control.

All recombinant proteins were dialyzed into TBS buffer and their concentrations were determined by measuring the OD at 280 nm.

Dot blot analysis:

The nitrocellulose membrane was spotted by serial dilution of the five recombinant MASP-2 polypeptides (Fig. 13) (and thioredoxin polypeptide as negative control for the CCPII-serine protease polypeptide) described above. The amount of protein spotted was in the range of 100 ng to 6.4 pg at the 5-fold stage. In later experiments, the amount of protein spotted was again in the range of 50 ng to 16 pg at 5-fold stage. The membranes were blocked with TBS solution (blocking buffer) of 5% skimmed milk powder and then incubated with a blocking buffer (containing 5.0 mM Ca2 +) of 1.0 μg / ml anti-MASP-2 Fab2s. Bound Fab2 was detected using HRP-conjugated anti-human Fab (AbD / Serotec; 1 / 10,000 dilution) and ECL detection kit (Amersham).

One membrane was incubated with polyclonal levit-anti-human MASP-2 Ab as a positive control (Stover et al., J Immunol 163: 6848-59 (1999)). In this case, bound Ab was detected using HRP-conjugated goat anti-rabbit IgG (Dako; 1/2, 000 dilution).

MASP-2 binding assay

ELISA plates were coated overnight at 4 占 폚 with 1.0 占 퐂 / well of a solution of recombinant MASP-2A or CUBI-II polypeptide in carbonate buffer (pH 9.0). Wells were blocked with 1% BSA TBS solution and serial dilutions of anti-MAST-2 Fab2 were added to 5.0 mM Ca2 + containing TBS. Plates were incubated for 1 hour at room temperature. After washing three times with TBS / tween / Ca2 +, a TBS / Ca2 + solution of HRP-conjugated anti-human Fab (AbD / Serotec) diluted 1 / 10,000 was added and the plate was further incubated at room temperature for 1 hour. Bound antibodies were detected using a TMB peroxidase substrate kit (Biorad).

result:

The results of dot blot analysis demonstrating the reaction of Fab2 with various MASP-2 polypeptides are shown in Table 7 below. The numerical values in Table 7 represent the amount of pointed protein needed to represent half the signal intensity. As shown, all of the polypeptides (as an example of thioredoxin fusion partners only) were recognized by positive control Ab (polyclonal anti-human MASP-2 serum, rinsed in rebit).

Table 7: Reaction of various recombinant MASP-2 polypeptides on dot blots Fab antibody # MASP-2A CUBI-II CUB / EGF-like CCPII-SP Thioredoxin 40 0.16 ng NR NR 0.8 ng NR 41 0.16 ng NR NR 0.8 ng NR 11 0.16 ng NR NR 0.8 ng NR 49 0.16 ng NR NR > 20 ng NR 52 0.16 ng NR NR 0.8 ng NR 57 0.032 ng NR NR NR NR 58 0.4 ng NR NR 2.0 ng NR 60 0.4 ng 0.4 ng NR NR NR 63 0.4 ng NR NR 2.0 ng NR 66 0.4 ng NR NR 2.0 ng NR 67 0.4 ng NR NR 2.0 ng NR 71 0.4 ng NR NR 2.0 ng NR 81 0.4 ng NR NR 2.0 ng NR 86 0.4 ng NR NR 10 ng NR 87 0.4 ng NR NR 2.0 ng NR Positive control <0.032 ng 0.16 ng 0.16 ng <0.032 ng NR NR = No reaction. Positive control antibody is a polyclonal anti-human MASP-2 serum, rinsed in rev.

All Fab2 reacted with MASP-2A and MASP-2K (data not shown). The majority of Fab2 recognized the CCPII-SP polypeptide but not the N-terminal fragment. The two exceptions were Fab2 # 60 and Fab2 # 57. Fab2 # 60 recognized the MASP-2A and CUBI-II fragments, but did not recognize the CUBI / EGF-like polypeptide or CCPII-SP polypeptide, indicating that it is an epitope in CUBII, or in the spanning CUBII and EGF- Lt; / RTI &gt; Fab2 # 57 recognizes MASP-2A but does not recognize all MASP-2 fragments tested, indicating that perhaps this Fab2 recognizes an epitope in CCP1. Fab2 # 40 and # 49 bind only to complete MASP-2A. In the ELISA binding assay (Figure 14), Fab2 # 60 also bound to the CUBI-II polypeptide, even though it had an apparently low affinity.

This finding demonstrates the identification of a unique blocking Fab2 for multiple regions of the MASP-2 protein.

Example 26

This example illustrates the analysis of MASP-2 - / - mice in a murine kidney ischemia / reperfusion model.

Background / Rationale : Ischemia-reperfusion (I / R) injury of the kidney at body temperature is associated with a number of clinical symptoms, such as circulatory hypovolemic shock, renal artery occlusion, and cross-clamp surgery.

Renal ischemia-reperfusion (VR) is an important cause of acute renal failure with a mortality rate of up to 50% (Levy et al., JAMA 275: 1489-94, 1996; Thadhani et al, N. Engl. : 1448-60, 1996). Post-transplant kidney failure is a common and threatening complication after renal transplantation (Nicholson et al., Kidney Int. 58: 2585-91, 2000). Effective treatment of kidney VR damage is currently absent, and is the only treatment available for hemodialysis. It is related to the pathophysiology of renal VR injury. Recent studies have found that the lectin pathway of complement activation plays an important role in the pathogenesis of renal VR injury (de Vries et al., Am. J. Path. 765: 1677-88, 2004).

Way:

MASP-2 (- / -) mice were generated as described in Example 1 and backcrossed with C57B1 / 6 for the production of at least 10 mice. The peritoneum of Hypnovel (6.64 mg / kg; Roche products Ltd. Welwyn Garden City, UK) was injected into 6 male MASP-2 (- / -) and 6 wild type (+ / + (Abbott Laboratories Ltd., Kent, UK) and subsequently anesthetized by inhalation. Isoflurane was chosen because it is an inhalation anesthetic with minimal hepatotoxicity; The concentrate was produced exactly and the animal quickly recovered even after prolonged anesthesia. Hip Nobel was given because it produces neuroleptic analgesic symptoms in animals, which means that a small amount of isoflurane is administered. To maintain a constant body temperature, I placed a warm pad under the animal. The central abdominal incision was then performed and the body cavity was opened using a retractor. The connective tissue was removed from above and below the renal veins, and the left and right renal arteries and kidney roots were clamped for 55 minutes with micro-aneurysm clamps. Ischemia of this period is based on previous studies of this laboratory (Zhou et al, J. Clin. Invest. 105: 1363-71 (2000)). In addition, the standard ischemic time of 55 minutes was selected after ischemia and found that 55 minutes gave reversible constant damage with a low mortality rate of 5% or less. After occlusion, 0.4 ml of warm saline (37 ° C) was covered over the abdomen for ischemic time. After removal of the micro-aneurysm clamp, blood renal blood flow and color change were observed in the kidney. An additional 0.4 ml of warm saline was placed in the abdominal cavity, the dog abdomen was sutured, and the animals were returned to us. Tail blood samples were collected 24 hours after clamp removal, and mice were sacrificed at 48 hours and additional blood samples were collected.

Evaluation of renal damage: Renal function was evaluated at 24 and 48 hours after reperfusion in 6 male MASP-2 (- / -) and 6 WT (+ / +) mice. Blood creatine measurements were measured by a mass spectrometer, which provides a reproducibility index of renal function (sensitivity <1.0 μmol / L). FIG. 15 shows the removal of blood urea nitrogen at 24 hours and 48 hours after reperfusion of the wild type C57B1 / 6 control and MASP-2 (- / -). As shown in Figure 15, MASP-2 (- / -) mice exhibit a significant reduction in the amount of blood elements at 24 and 48 hours compared to wild-type control mice, indicating a protective function against renal injury in the ischemic reperfusion injury model Effect.

Overall, increased blood elements appear in both WT (+ / +) and MASP-2 (- / -) mice at 24 and 48 hours after surgical surgery and ischemic injury. Blood element levels in non-ischemic WT (+ / +) surgical animals were determined separately at 5.8 mmol / L. Along with the data shown in FIG. 15, one MASP-2 (- / -) animal showed values of 6.8 and 9.6 mmol / L at 24 and 48 hours, respectively, indicating almost complete protection of ischemic injury. This animal was excluded from the analysis of the population as a potential anomaly, and does not exhibit ischemic damage here. Thus, the final analysis of FIG. 15 included 5 MASP-2 (- / -) mice and 6 WT (+ / +) mice and MASP-2 (- / -) mice at 24 and 48 hours Showing a statistically significant decrease in blood elements (Student t-test ρ <0.05). This finding implies that inhibition of MASP-2 activity is expected to have a protective or therapeutic effect of renal injury due to ischemic injury.

Example 27

This example illustrates the analysis of MASP-2 (- / -) mice in a mouse myocardial ischemia / reperfusion model.

Background / Rationale:

Mannose-binding lectin (MBL) is a circulating molecule that initiates complement activation in an immune complex-independent method that responds to a wide range of carbohydrate structures. This structure can be a component of the infectious agent or, in particular, changes the endogenous carbohydrate moiety within necrosis, carcinogenic or apoptotic cells. This type of cell death develops in reperfused myocardium, and complement activation seems to be extended to impairment prior to the zone present at the time when ischemia is terminated by reperfusion. Although there is clear evidence that complement activation exacerbates myocardial reperfusion, this activation mechanism is not well known, and inhibition of all known pathways appears to have unacceptable adverse effects. Recent studies indicate that MBL is involved rather than the classical pathway or alternative amplification loop (as defined by the present invention). This is because the infarct was reduced in MBL (A / C) - but not in C1q- and null mice (Walsh M.C. et al., Jourin Immunol. 775: 541-546 (2005)). However, such a mouse still retains circulating components, such as picoline A, that can activate the complement through the lectin pathway.

This study investigated the MASP-2 (- / -) mouse versus wild type (+ / +) control to determine whether MASP-2 (- / -) is less sensitive to myocardial ischemia and reperfusion injury. Local ischemia was applied to MASP-2 (- / -) mice and the infarct size was compared with their wild-type offspring.

METHODS: The following protocol is described in the aforementioned document Marber et al., J. CHn Invest. 95: 1446-1456 (1995). &Lt; / RTI &gt;

MASP-2 (- / -) mice were generated as described in Example 1 and backcrossed with C57B1 / 6 for the production of at least 10 mice. Seven MASP-2 (- / -) mice and seven wild type (+ / +) mice were anesthetized with ketamine / medetomidine (100 mg / kg and 0.2 mg / kg, respectively) It was laid down directly on the thermostatic thermal pad to keep it at ± 0.3 ° C. The animals were anesthetized with a respiratory rate of 110 / min and a ventilation rate of 225 μl / min (Hugo Sachs Elektronic MiniVent Type 845, Germany).

The fur was shaved and anterior exoskeletal incision was made from the left armpit to the dorsal surface. The pectoralis major was incised, resected to the sternal border, and moved to the underarm gorilla. The pectoralis muscle was resected to the cranial border and moved to the tail. Muscle was used as a muscle flap after occlusion of the coronary artery. Muscle and wall lateral pleura of the 5th intercostal space penetrated the border of the left lung with a slight twist in the middle, thus avoiding lung or heart damage. After the pleural penetration, the tweezers were carefully placed behind the pleura in the direction of the sternum to avoid contact with the heart, and the pleural and intercostal muscles were resected with a motor excision machine (Harvard Apparatus, UK). Special care was taken to prevent bleeding. Using the same technique, the thoracotomy was extended to the central axillary line. After removing ribs 4, the intercostal space was extended until the entire heart was exposed. Two small arterial clamps were used to open the pericardium, and the pericardium was moved slightly forward. The left anterior descending coronary artery (LAD) was exposed and passed under the LAD with an 8-0 monofilament suture and a round needle. The LAD ligature is located on the tail side of the upper part of the left atrium, and approximately 1/4 of the line extends from the atrioventricular ridge to the end of the left ventricle.

All experiments were performed blindly and the experimenter did not recognize the genotype of each animal. After instrument manipulation and surgical procedures, mice were given an equilibration period of 15 minutes. The mice were then subjected to a 30-minute coronary occlusion and 120 minutes of reperfusion.

Cardiac artery occlusion and perfusion model

Cardiac artery occlusion was achieved using the weight-bearing system described above (Eckle et al., Am J Physiol Heart Circ Physiol 291: H2533-H2540, 2006). The two ends of the monofilament ligation were passed through a 2 mm long piece of polythene PE-10 tubing and attached to the 5-0 suture length using cyanoacrylate glue. The suture was connected to two horizontally mounted movable rods and each mass of 1 g was attached to both ends of the suture. As the load is increased, the mass is further suspended and the suture area undergoes pressure to receive the defined LAD and constant pressure. LAD occlusion is evidenced by the vitality of the area of risk, and the color of the LAD perfusion area changes from bright red to purple, which means the interruption of blood flow. The reperfusion is achieved by lowering the load until it comes to the mass add-treatment pad and ligature pressure is relieved. Reperfusion is evidenced by the same three criteria used to establish obstruction. Mice are excluded from further analysis if all three criteria are inconsistent at the onset of coronary occlusion or 15 minutes of reperfusion each. During coronary occlusion, the temperature and humidity of the heart surface is covered by a pectoralis muscle flap and maintained by an open thoracotomy with 0.9% saline wet gauze.

Measurement of myocardial infarct size :

Infarct size (INF) and hazard area (AAR) are measured by planometry. 500 LU. After iv injection of the hairpin, LAD was re-occluded and 300 μl of 5% (w / vol) Evans blue (Sigma-Aldrich, Poole, UK) was slowly injected into the carotid artery to demonstrate the AAR. This causes dye to enter the non-ischemic area of the left ventricle, and the ischemic AAR remains unstained. After cervical dislocation, mice are sacrificed and the heart is quickly removed. The heart is cooled in ice, mounted on a block of 5% agarose, and then cut into eight pieces of 800 μm thick cross-sections. All slices at 37 ℃ for 20 _{minutes, 0.1 M Na 2 HPO 4 /} NaH 2 PO 4 buffer with 3% 2,3,5-triphenyl tetrazolium chloride (Sigma Aldrich, Poole, UK) dissolved in (pH 7.4) Lt; / RTI &gt; The slices were fixed overnight in 10% formaldehyde. The slice was placed between two cover slits and the side of each slice was digitally imaged using a high resolution optical scanner. Digital images were analyzed using SigmaScan software (SPSS, US). The size of the infarcted area (pale), left ventricle (LV) critical area (red) and normal perfusion LV area (blue) were outlined in each section by confirmation of appearance color and color boundaries. The area was quantified on both sides of each slice and averaged by the investigator. The infarct size was calculated as a percentage of the hazardous area of each animal.

Results: The size of the infarcted area (pale), LV hazard area (red) and steady flow LV area (blue) were outlined in each section by confirmation of appearance color and color boundaries. The area was quantified on both sides of each slice and averaged by the investigator. The infarct size was calculated as a percentage of the hazardous area of each animal. 16A shows the evaluation of 7 WT (+ / +) mice and 7 MASP-2 (- / -) mice to determine the infarct size of these after performing the coronary artery occlusion and reperfusion technique described above . As shown in FIG. 16A, MASP-2 (- / -) mice exhibit a statistically significant decrease in infarct size (p <0.05) compared to wild type (+ / +) mice, It means a protective myocardial effect. Figure 16B shows the distribution of the individual animals tested, which shows a clear protective effect of MASP-2 (- / -) mice.

Example 28

This example illustrates the results of MASP-2 - / - in the murine macular degeneration model.

Background / Rationale : Age-related macular degeneration (AMD) is a major cause of loss of vision after age 55 in industrialized societies. AMD occurs in two major forms: neovascular (wet) AMD and atrophic (dry) AMD. The neovascular (wet) form is responsible for 90% of the severe vision loss associated with AMD, even though only ~ 20% of AMD patients are wet. Clinical hallmarks of AMD include multiple drusen, mental atrophy, and colloid neovascularization (CNV). In December 2004, the FDA approved a new class of ophthalmic drugs that specifically targeted and blocked Macugen (pegaptanib) to the effects of vascular endothelial growth factor (VEGF) for the treatment of wet (neovascular) type AMD (Ng et al., Nat Rev. Drug Discov 5: 123-32 (2006)). Although makugen has shown promising therapeutic effects in patients with AMD, there are attempts to develop additional therapies for this complex disease. Multidisciplinary, non-existent studies of AMD play a central role in complement activation in AMD pathogenesis. The pathogenesis of colloid neovascularization (CNV), the most severe form of AMD, is related to the activation of the complement pathway.

Twenty-five years ago, Ryan described a model of laser-induced damage of CNV in animals (Ryan, S. J., Tr. Am. Opth. Soc. LXXVII-JOl-145, 1979). Although this model was originally developed using regus monkeys, the same technique has been used to develop similar CNV models of various study animals, such as mice (Tobe et al., Am. J. Pathol. 753: 1641-46, 1998). In this model, laser photocoagulation was used to rupture the brunch membrane, which is the formation of a CNV-like membrane. The laser-induced model characterizes a number of important human symptoms (Ambati et al., Survey Ophthalmology 48: 257-293, 2003). The laser-induced mouse model is now well established and used as the experimental base for large-scale research projects. In general, laser-induced models are recognized to share sufficient biological similarity with human CNVs, and preclinical studies of the onset mechanism using this model and drug inhibition are related to CNV in humans.

Way:

A MASP-2 - / - mice were generated as described in Example 1 and backcrossed with C57B1 / 6 for the production of 10 mice. The present study compared the results when MASP-2 (- / -) and MASP-2 (+ / +) male mice were evaluated within the course of laser-induced CNV, Accelerated neovascular AMD models focused on the volume of laser-induced CNV by laser confocal microscopy scanning as a measure of epithelial (RPE) / choroidal tissue damage and VEGF levels, and potential angiogenic factors associated with CNV.

Induction of colloid neovascularization (CNV): Experiments by single entity labeled drug group alignment Laser light coagulation (532 nm, 200 mW, 100 ms, 75 μm; Oculight GL, Iridex, Mountain View, CA). A laser spot was applied around the optic nerve in a standardized manner using a cover slip as a split lamp delivery system and a contact lens. The morphological endpoint of the laser injury was the appearance of foam cavitation, which appears to be related to the destruction of the bruch membrane. The detailed method and the estimated end point are as follows.

Fluorescein angiography: Fluorescein angiography was performed with a camera and imaging system (TRC 50 IA camera; ImageNet 2.01 System; Topcon, Paramus, NJ) one week after laser light fixation. The photographs were captured with a 20-D lens in contact with the base camera lens after intraperitoneal injection of 0.1 ml of 2.5% sodium fluorescein. The retina specialist was not involved in laser photocoagulation or angiography, which evaluated fluorescein angiography in a single setting within the indicated method.

Volume of colloid neovascularization (CNV) : One week after laser injury, the eyes were fixed with 4% paraformaldehyde at 4 ° C for 30 min. Washing cups were obtained by removing the anterior part, washed three times with PBS, and dehydrated and rehydrated through the methanol series. Galactose residues on the surface of endothelial cells after blocking with buffer (containing PBS, 1% bovine serum albumin and 0.5% Triton X-100) for 30 minutes at room temperature twice, and the murine vasculature (Vector laboratories, Burlingame, Calif.) Diluted in PBS containing 0.2% BSA and 0.1% Triton X-100. After washing twice with PBS containing 0.1% Triton X-100, the neurosensory retina was slowly removed and removed from the optic nerve. Four relaxation radical incisions were made, and the remaining RPE-choroid-sclera complex was placed in an Immu-Mount Vectashield Mounting Medium (Vector Laboratories) and covered.

Plaques were scanned with a scanning laser confocal microscope (TCS SP; Leica, Heidelberg, Germany). The blood vessels were visualized by excitation with a blue argon wavelength (488 nm), capturing emissions between 515 and 545 nm. A 40X oil-immersion object was used for imaging studies. Horizontal optical sections (1 μm steps) were obtained from the RPE-choroid-scleral complex surface. The deepest focal plane that can identify the surrounding colloid vascular network connected to the lesion was determined to be at the bottom of the lesion. The appearance of all blood vessels and this reference plane within the laser-targeting area was determined by CNV. The images of each section were stored digitally. The CNV-fluorescent region was measured by computer image analysis with a microscope software (TCS SP; Leica). The sum of the fluorescent regions in each horizontal section was used as an index of CNV volume. Imaging was performed by an operator designated as a treatment group.

Laser lesion development Since the likelihood of CNV is affected by their population (mouse, eye, and laser spot), the mean lesion volume was compared using a linear blending model of split-plots repeat-measurement design. The total plot factor is the hereditary group of animals, where the split plot factor is the eye. Statistical significance was determined at the 0.05 level. Post hoc comparison averages are constructed with this Bonferroni adjustment for multiple comparisons.

VEGF ELISA . 12, three days after injury by the laser spot, the lysis buffer RPE- choroid complex (20 mM imidazole HCl, 10 mM KCl, 1 mM MgCL 2, 10 mM EGTA, 1% Triton X-100, 10 mM NaF, 1 mM Na molybdate, and 1 mM EDTA, protease inhibitor) for 15 minutes on ice. VEGF protein levels in the supernatants were measured at 450-570 nm (Emax; Molecular Devices, Sunnyvale, Calif.) With an ELISA kit (R & D Systems, Minneapolis, MN) that recognizes all splice variants and normalized to total protein. Two-bead measurements were performed by an experimenter who was not involved in photocoagulation, imaging, or angiography. The number of VEGFs is represented by at least three independent tests of mean +/- SEM and compared using the Mann-Whitney U test. The null hypothesis was rejected at P <0.05.

result:

Assessment of VEGF levels:

Figure 17A shows VEGF protein levels in the RPE-choroid complex isolated from C57B16 wild type and MASP-2 (- / -) mice at day 0 of the experiment. As shown in Figure 17A, evaluation of the VEGF level indicates that the basal level of VEGF in the MASP-2 (- / -) mice is reduced compared to the C57bl wild type control mice. Figure 17B shows the measured levels of VEGF protein after 3 days of laser induced injury. As shown in Figure 17B, VEGF levels were significantly increased in wild type (+ / +) mice at 3 days after laser induced injury, consistent with published studies (Nozaki et al, Proc. Natl. Acad. ScL USA 103: 2328-33 (2006)). However, it exhibits significantly lower levels of VEGF in MASP-2 (- / -) mice.

Evaluation of colloid neovascularization (CNV) :

With the reduction of VEGF levels after laser-induced macular degeneration, the CNV area was determined before and after laser injury. Figure 18 shows CNV volumes measured in C57bl wild-type mice and MASP-2 (- / -) mice 7 days after laser induced injury. As shown in FIG. 18, MASP-2 (- / -) mice exhibited a 30% reduction in CNV area after 7 days of laser induced injury compared to wild type control mice. These findings indicate that VEGF and CNV are reduced in MASP (- / -) mice compared to wild type (+ / +) control, and MASP-2 blockade by inhibitor prevents or treats macular degeneration .

Example 29

This example illustrates the results of MASP-2 (- / -) in a murine monoclonal antibody-induced rheumatoid arthritis model.

Background / Rationale : The most commonly used rheumatoid arthritis (RA) animal model is collagen-induced arthritis (CIA) (Linton and Morgan, Mol. Immunol. 36: 905-14, 1999). Collagen type II (CII) is one of the major components of joint matrix proteins, and immunization by natural CII in adjuvants induces autoimmune polyarthritis by CII cross-reactive autoimmune reactions in articular cartilage. In RA, susceptibility to CIA is associated with the expression of certain Class II MHC homologs. Some mouse strains, including the C57B1 / 6 strain, are resistant to classical CIAs and lack the proper MHC haplotype, thus not producing the anti-CII antibody titers. However, it has been found that cocktails of four specific mAb to type II collagen can be induced in all mouse strains by iv or ip administration to the mice. These arthritis-inducing monoclonal antibodies can be obtained (Chondrex, Inc., Redmond, WA). The passive model of this CIA reported that C57B1 / 6 mouse strains were used in a number of recent studies (Kagari et al., J. Immunol. 169: 1459-66, 2002. Kato et al., J. Rheumatol. : 241-55, 2003; Banda et al, J. Immunol. 177: 1904-12, 2006). Subsequent studies compared the sensitivities of wild type (+ / +) (WT) and MASP-2 (- / -) mice to arthritis development using a CIA passive transfer model, both of which share a C57B1 / 6 genetic background do.

Way:

Animal: A MASP-2 (- / -) mice were produced as described in Example 1 and backcrossed with C57B1 / 6 for the production of 10 mice. 14 male and female C57BL / 6 wild-type mice at 7-8 weeks of age and 10 male and female MASP-2 (- / -) and wild type (+ / +) C57B1 / 6 mice were used in this study. Twenty mice were injected with a monoclonal antibody cocktail (group of 10 mice) to obtain 20 definite reactants. Animals (10 / group) were fed with 5 birds per U. Five animals were acclimated seven days before the start of the study.

Monoclonal antibody cocktail (Chondrex, Redmond WA) (5 mg) was intravenously injected into the mice on days 0 and 1 of the experiment. The test agent was Chondrex monoclonal antibody + LPS. On day 2 of the experiment, mice were ip dosed with LPS. Mice were weighed at 0, 2, 4, 6, 8, 10, 12 days and 14 days before termination. On day 14, the mice were anesthetized with isoflurane and finally the serum was bled. After blood collection, mice were euthanized, and the front and hind legs were removed to the knees and placed in formalin for further processing.

Treatment group:

Group 1 (control): 4 mice C57 / BL / 6 WT (+ / +) week;

Group 2 (test group): 10 mice C57 / BL / 6 WT (+ / +) mice (mAb cocktail + LPS); And

3 mice (test group): 10 mice C57 / BL / MASP-2KO / 6Ai (- / -) mice (mAb cocktail + LPS)

Clinical arthritis scores were assessed daily with the following scoring system:

0 = normal; 1 = 1 back or paw joint effect; 2 = 2 back or paw joint effects; 3 = 3 back or paw joint effects; 4 = moderate (swelling of erythema and swelling, or 4-toe joint effects); 5 = severe (diffuse erythema and severe swelling of the feet, no toe flow)

result:

Figure 19 shows group data illustrating the average daily clinical arthritis score for up to two weeks. Clinical arthritis scores were not observed in the control group without CoL2 MoAb treatment. MASP (- / -) mice received a low clinical arthritis score on days 9-14. The overall clinical arthritis score is the area under the curve analysis (AUC), which shows a 21% reduction in MASP-2 (- / -) compared to WT (+ / +) mice. However, the C57B16 mouse background described above does not provide a robust total arthritic clinical score. Due to the small incidence and the size of the population, even in the case of positive trends, the data provided only trends (p = 0.1) and were not statistically significant at the p < 0.05 level. Additional animals in the treatment group are needed to indicate statistical significance. Due to the reduced incidence of arthritis, the score of the developed foot was evaluated with respect to severity. A single occurrence of three or more clinical arthritic scores was not found in the MASP-2 (- / -) mice and was observed in 30% of WT (+ / +) mice and additional suggestions were (1) May be involved in complement pathway activation and (2) blockade of MASP-2 may be beneficial to arthritis.

Example 30

This example demonstrates that miniature mannose-binding lectin-binding proteins (Map 19 or sMAP) are inhibitors of MASP-2 dependent complement activation.

Background / Rationale:

summary:

Mannose-binding lectins (MBL) and picolines are pattern-recognition proteins that activate congenital immunity and trigger activation of the lectin complement pathway through MBL-induced serine proteases (MASPs). In activation of the lectin pathway, MASP-2 degrades C4 and C2. Small MBL-associated protein (sMAP), truncated MASP-2 binds to the MBL / picoline-MASP complex. To clarify the role of sMAP, sMAP-specific exon targeted destruction was performed to produce sMAP-deficient (sMAP - / -) mice. By gene disruption, expression levels of MASP-2 in sMAP - / - mice decreased. When recombinant sMAP (rsMAP) and recombinant MASP-2 (rMASP-2) were reconstituted MBL-MASP-sMAP complex in deficient serum, the binding of these recombinants to MBL was competitive and the binding of MBL-MASP-sMAP complex C4 The cleavage activity is restored by the addition of rMASP-2, where the addition of rsMAP reduces activity. Thus, MASP-2 is essential for C4 activation, and sMAP plays a modulatory role in the activation of the lectin pathway.

Introduction:

The complement system mediates the chain reaction of proteolysis and the assembly of protein complexes and plays a major role in biological defense as part of both the innate and acquired immune systems. The mammalian complement system consists of three activation pathways, a classical pathway, an alternative pathway, and a lectin pathway (Fujita, Nat. Rev. Immunol. 2: 346-353 (2002); Walport, N Engl J Med 344: 1058- 1066 (2001)). The lectin pathway provides a primary line of defense against invading pathogens. Mannose-binding lectin (MBL) and picoline, the pathogen recognition components of this pathway, bind to a carbohydrate array on the surface of bacteria, viruses, and parasites and activate MBL-coupled serum proteases (MASPs) . The importance of the lectin pathway for congenital immune defense has been pointed out in a number of clinical studies of the deficiency of MBL with increased susceptibility to various infectious diseases in infancy, especially before the acquired immune system was established (Jack et al. , Summer et al, Lancet 345: 886-889 (1995); Super et al., Lancet 2: 1236 (1995); Immunol Rev 180: 86-99 (2001); Neth et al. Infect Immun 68: 688-693 -1239 (1989)). However, the lectin pathway also contributes to undesired complement activation, which is involved in a number of pathological conditions such as inflammation and tissue damage in ischemia / perfusion damage of the heart and kidney (de Vries et al., Am J Pathol 165 Walsh et al., J Immunol 175: 541-546 (2003); Jordan et al., Circulation 104: 1413-1418 (2001); (2005)).

As described above, the lectin pathway is involved in carbohydrate recognition by MBL and picoline (Fujita et al, Immunol Rev 198: 185-202 (2004); Holmskov et al., Annu Rev Immunol 21: 547-578 (2003) (Matsushita and Fujita, J Exp Med 176: 1497-1502 (1992); Sato et al, lnt Immunol 6: 665-669 (1994); Matsushita and Fujita, Immunobiology 205: 490-497 MASP-2 (Thiel et al., Nature 386: 506-510 (1997), MASP-3 (Dahl et al, Immunity 15: 127- 135 (2001), and MASP-2 cleaved proteins (small MBL-associated protein: sMAP or MApl9) (Stover et al, J Immunol 162: 3481-3490 (1999); Takahashi et al, lnt Immunol 11: 8590863 1999). MASP family members consist of six domains: two C1r / C1s / Uegf / osteogenic protein (CUB) domains, epidermal growth factor (EGF) -like domains, two complement control proteins CCP) or short consensus repetition (SCR) MASP-2 and sMAP are generated by alternative splicing from a single structural gene, sMAP is the first CUB (CUBI), and the serine protease domain (Matsushita et al, Curr Opin Immunol 10: 29-35 Domain, the EGF-like domain, and the extra 4 amino acids at the C-terminal end encoded by the sMAP-specific exon. MASP-1 and MASP-3 can be generated from a single gene by alternative splicing (Schwaeble et al, Immunobiology 205: 455-466 (2002). When MBL and picoline bind to the carbohydrate on the surface of the microbe, the enzyme precursor MASP degrades between the second CCP and the protease domain to form two polypeptides referred to as heavy chain (H) - and light chain (L) And becomes the configured active form. Thus, the proteolytic activity of the complement component is obtained. Accumulated evidence indicates that MASP-2 degrades C4 and C2 to induce the formation of C3 converting enzyme (C4bC2a) (Matsushita et al, J. Immunol 165: 2637-2642 (2000)). We suggest that MASP-1 directly degrades C3 and subsequently activates the amplification loop (Matsushita and Fujita, Immunobiology 194: 443-448 (1995), but this function is controversial (Ambrus et al , J. Immunol 170: 1374-1382 (2003). Although MASP-3 contains serine protease domains in the L-chain and exerts their proteolytic activity on synthetic substrates (Zundel et al, J Immunol 172: 4342 -4350 (2004), their physiological substrate was not identified. The function of sMAP lacking the serine protease domain is still unknown.

In the present study, to clarify the role of sMAP in activating the lectin complement pathway, we disrupted the sMAP-specific exon encoding the 4 amino acid residues (EQSL) at the C-terminus of sMAP, - mice. We first reported the ability of sMAP to activate the down-regulated lectin pathway.

Materials and methods

mouse

The targeting vector is constructed to include a neomycin resistance gene cassette in place of exons 1-4 and exons 6 of the 129 / Sv mouse MASP-2 gene and exon 5 (FIG. 20A). The DT-A gene is inserted at the 3 'end of the vector and the three lox p sites are inserted to perform conditional targeting in the future to remove the neomycin cassette and promoter regions. The targeting vector was electroporated into 129 / Sv ES cells. Targeted ES clones were microinjected into C57BL / 6J cells implanted into the uterus of the parental ICR matrix. Male chimeric mice were crossed with female C57BL / 6J mice to produce heterozygous (+/-) mice. Heterozygous (+/-) mice were selected by tail DNA Southern blot analysis digested with BamH I using probes (FIG. 20A). Southern blot analysis shows 6.5-kbp and 11-kbp bands in the DNA of heterozygous (+/-) mice (Fig. 20B). Heterozygous (+/-) mice were backcrossed with C57BL / 6J mice. To obtain homozygous (- / -) mice, heterozygous (+/-) mice were crossbred. Homozygous (- / -) mice (C57BL / 6J background) were identified by PCR-based genotyping of tail DNA. PCR analysis was performed using a mixture of exon 4-specific and neo gene-specific sense primers and exon 6-specific antisense primers. DNA of homozygous (- / -) mice showed a single 1.8-kbp band (Fig. 20C). In all experiments, 8-12 week old mice were used according to the animal study guide of Fukushima Medical University.

Northern blot analysis

(A) + RNA (1 ㎍) between wild type (+ / +) and homozygous (- / -) mice was separated by electrophoresis and the nylon membrane was moved to obtain sMAP, MASP-2 H- L-chain, or neo gene-specific 32P-labeled cDNA probe. The same membrane was stripped and remarried with a specific probe to glyceraldehyde-3-phosphate dehydrogenase (GAPDH).

Quantitative RT-PCR

Real-time PCR was performed with a LightCycler system (Roche Diagnostics). CDNA synthesized in 60 ng poly (A) + RNA between wild type (+ / +) and homozygous (- / -) mice was used as template for real-time PCR, and MASP-2 H- and L- The cDNA fragment was amplified and monitored.

Immune blotting

The sample was electrophoresed on a 10 or 12% SDS-polyacrylamide gel under reducing conditions and proteins were transferred to a polyvinylidene difluoride (PVDF) membrane. Membrane proteins are detected with anti-MASP-2 / sMAP antiserum generated against peptides of anti-M ASP-I antiserum or MASP-2 H chain generated against the L-chain of MASP-1.

Detection of MASPs and sMAP in MBL-MASP-sMAP complex

Mouse serum (20 μl) was added to 480 μl of TBS-Ca 2+ buffer (20 mM Tris-HCl, pH 7.4, 0.15 M NaCl, and 5 mM CaCl 2) containing 0.1% (w / v) BSA ₂ ) and incubated for 30 min at 40 ° C in a TBS-Ca2 + / BSA buffer solution of 40% 50% mannan-agarose gel slurry (Sigma-Aldrich, St. Louis, Mo.). After incubation, each gel was washed with TBS-Ca2 + buffer and sample buffer for SDS-PAGE was added to the gel. The gel was boiled, SDS-PAGE was applied to the supernatant, and MASP-1, MASP-2, and sMAP in the MBL complex were detected by immunoblotting.

C4 attachment measurement method

Mouse serum is diluted to 100 μl with TBS-Ca 2+ / BSA buffer. The diluted samples were added to metered-coated microtiter wells and incubated at room temperature for 30 minutes. The wells were washed with cold wash buffer (TBS-Ca2 + buffer, containing 0.05% (v / v) Tween 20). After washing, human C4 was added to each well and incubated on ice for 30 minutes. The wells were washed with cold wash buffer and HRP-conjugated anti-human C4 polyclonal antibody (Biogeneis, Poole, England) was added to each well. After 30 minutes of incubation at 37 ° C, the wells were washed with wash buffer and 3,3 ', 5,5'-tetramethylbenzidine (TMB) solution was added to each well. After development, 1 MH ₃ PO ₄ was added and the absorbance at 450 nm was measured.

C3 attachment measurement method

Mouse serum was diluted to 100 μl with BSA buffer (4 mM barbiturate, 145 mM NaCl, 2 mM CaCl ₂ , and 1 mM MgCl ₂ , pH 7.4) containing 0.1% (w / v) HSA. The diluted samples were added to metered-coated microtiter wells and incubated at 37 占 for 1 hour. The wells were washed with wash buffer. After washing, HRP-conjugated anti-human C3c polyclonal antibody (Dako, Glostrup, Denmark) was added to each well. After 1 hour incubation at room temperature, the wells were washed with wash buffer and TMB solution was added to each well. The color was measured as described above.

Recombination

Recombinant mouse sMAP (rsMAP), rMASP-2, and an inactive mouse MASP-2 mutant (MASP-2i) in which the active-site serine residue in the serine protease domain was replaced with an alanine residue were prepared as previously described (Iwaki and Fujita, 2005).

Reconstitution of MBL-MASP-sMAP complex

Allogeneic (- / -) mouse sera (20 μl) and various amounts of MASP-2i and / or rsMAP were incubated with ice-cold overnight in 40 μl of TBS-Ca 2+ buffer. This mixture was incubated with a meta-agarose gel slurry and MASP-2i and rsMAP in the MBL-MASP complex bound to the gel were detected as described in "Detection of MASPs and sMAP in MBL-MASP-sMAP complex".

Reconstitution of C4 adherent activity

Allogeneic (- / -) mouse sera (0.5 μl) and various amounts of rMASP-2 and / or rsMAP were incubated with ice-cold overnight in a total volume of 20 μl of TBS-Ca2 +. The mixture was diluted with 80 [mu] l of TBS-Ca2 + / BSA buffer and added to the mann-coated wells. All subsequent steps were performed as described in "C4 Attachment Assay ".

result

Figure 20: Targeting disruption of the sMAP gene. (A) A partial restriction map of the MASP-2 / sMAP gene, the targeting vector, and the targeting homolog. Replace the sMAP-specific exon (exon 5) with a neo gene cassette. (B) Genomic DNA Southern blot analysis of offspring induced by crossing of male chimeric mice and female C57BL / 6J mice. The tail DNA is digested with BamHI and hybridized with the probe lacking (A). The 11-kbp band is derived from the wild-type homolog and the 6.5-kbp band is derived from the targeting homolog. (C) PCR genotype analysis. Tail DNA was analyzed using a mixture of exon 4-specific and neo gene-specific sense primers and exon 6-specific antisense primers. The 2.5-kbp band was obtained from the wild-type homolog and the 1.8-kb band was obtained from the targeting homolog.

Figure 21: Expression of sMAP and MASP-2 mRNAs in homozygous (- / -) mice. (A) Northern blot analysis. (A) + RNAs between wild type (+ / +) and homozygous (- / -) mice were electrophoresed, transferred to nylon membrane, and sMAP, MASP-2 H-chain, MASP- Or a 32P-labeled probe specific for the neo gene. Neo (2.2 kb) specific bands were observed in homozygous (- / -) mice. (B) Quantitative RT-PCR. MASP-2 H- and L-chain and sMAP cDNA fragments were amplified by real-time PCR in a LightCycler instrument (Roche Diagnostics). CDNAs synthesized from poly (A) + RNAs between wild type (+ / +) and homozygous (- / -) mice were used as templates. The data shown are the average of the two experiments.

Figure 22: MASP-2 deficiency in homozygous (- / -) mouse serum. (A) Immunoblotting of MASP-2 and sMAP in mouse serum. Anti-MASP-2 / sMAP antiserum was detected by immunoblotting in wild-type (+ / +) or homozygous (- / -) mouse serum (2 μl). (B) MASPs and sMAP detection in MBL-MASP-sMAP complex. Mouse sera were incubated with silver mannan-agarose gels and detected as described in the sMAP, MASP-1, and MASP-2 materials and methods in the MBL complex bound to the gel.

Figure 23: Reduced cleavage of C4 and C3 in homozygous (- / -) mouse serum. (A) met-C4 attachment on the coated well. Mouse serum is diluted 2-fold and incubated in mannan-coated wells at room temperature for 30 minutes. After well washing, human C4 was added to each well and incubated on ice for 30 minutes. The amount of human C4 attached to the wells was measured using an HRP-conjugated anti-human C4 polyclonal antibody. (B) Met-adhesion of C3 on the cladding well. Diluted mouse serum was added to the confluent-coated wells and incubated for 1 hour at 37 degrees. Immobilization of C3 on the wells was detected as an HRP-conjugated anti-human C3c polyclonal antibody.

Figure 24: Competitive binding of sMAP and MASP-2 to MBL. (A) Reconstitution of MBL-MASP-sMAP complex in homozygous (- / -) mouse serum. MASP-2i and / or rsMAP (4 ㎍) were incubated with homozygous (- / -) mouse serum (20.). This mixture was further incubated with mannan-agarose gel, and rsMAP and MASP-2i on the gel bound fraction were detected by immunoblotting. (B) Various amounts of MASP-2i (0-5 ㎍) and a certain amount of rsMAP (5 ㎍) were cultured with homozygous (- / -) mouse serum (20 ㎕) and further incubated with mannan-agarose gel. (C) A certain amount of MASP-2i (0.5 μg) and various amounts of rsMAP (0-20 μg) were incubated with homozygous (- / -) mouse serum (20 μl). (D) Various amounts of rsMAP (0-20 ㎍) were incubated with wild type (+ / +) mouse serum (20 ㎕).

Figure 25: Recovery of C4 adherence activity by addition of rMASP-2. Various amounts of rsMAP (0-5 μg) (A) or rMASP-2 (0-1.5 μg) (B) were mixed with 0.5 μl of homozygous (- / -) mouse serum in a total volume of 20 μl of TBS-Ca 2+ buffer And incubated overnight on ice. The mixture was then diluted with 80 [mu] l TBS-Ca2 + / BSA buffer, added to the mannan-coated wells, and the amount of C4 adhered to the wells was measured.

Figure 26: Reduction of C4 adherence activity by addition of sMAP. (A) rMASP-2 (1 μg) and various amounts of rsMAP (0-0.5 μg) were incubated with 0.5 μl of homozygous (- / -) mouse serum. (B) rsMAP (0-0.7 [mu] g) was incubated with wild-type serum (0.5 [mu] l) and the C4 attached to the mannan-coated wells Was measured.

result:

Expression of sMAP and MASP-2 in homozygous (- / -) mice

To clarify the role of sMAP in vivo, we established gene targeted mice lacking sMAP. The targeting vector was constructed to replace the sMAP specific exon (exon 5) with a neomycin resistant gene cassette (Fig. 20A). Positive ES clones were injected into C57BL / 6 blastocysts and Shijo chimeras were cultured in C57BL / 6J female. Tail DNA Southern blot analysis of the guinea pig-color puff shows bacterial transfer of the targeting homologue (Figure 20B). Heterozygous (+/-) mice are selected by Southern blot analysis of tail DNA digested with BamH I using the probe of FIG. 20A. Southern blot analysis shows the 6.5-kbp and 11-kbp bands in the DNA of heterozygous (+/-) mice (Figure 20B). Heterozygous (+/-) mice were backcrossed with C57BL / 6J mice. To obtain homozygous (- / -) mice, heterozygous (+/-) mice were heterozygous. Homozygous (- / -) mice (C57BL / 6J background) were identified by PCR-based genotyping of tail DNA and yielded a single 1.8-kbp band (Figure 20C).

Allogeneic (- / -) mice developed normally and did not show a significant difference in weight with wild type (+ / +) mice. There was no morphological difference between them. In Northern blot analysis, sMAP-specific probes detected a single 0.9-kb band in the wild type (+ / +) mouse, whereas no specific band was detected in the homozygous (- / -) mouse 21A). Several specific bands were detected in wild type (+ / +) mice as described above (Stover et al, 1999), when using a probe specific for MASP-2 H- or L- -Specific probe also detected the sMAP specific-band. However, within the homozygous (- / -) mouse, the corresponding band was very weak and several additional bands were detected. We also performed quantitative RT-PCR analysis to examine the expression levels of sMAP and MASP-2 mRNAs. In allogeneic (- / -) mice, expression of sMAP mRNA was completely blocked and MASP-2 was also significantly reduced: about 2% of the wild type (+ / +) mice in the H- and L- (Fig. 21B). Additionally, expression of MASP-2 was examined at the protein level. Both sMAP and MASP-2 were not detectable by homologous (- / -) mouse sera by immunoblotting (Fig. 22A). All of sMAP and MASP-2 could not be detected in the gel-bound fraction (Fig. 22B), even though MASP-1 was detected in the complex after incubation with agarose gels of homozygous (- / -) mouse sera.

 Decomposition activity of C4 and C3 through the lectin pathway in homozygous (- / -) mouse serum

When the allogeneic (- / -) mouse serum was cultured in a confluent-coated well, the amount of human C4 adhered to the well was about 20% of the diluted normal serum in the range of 1/400 to 1/50 (Figure 23A) . We investigated the C3 attachment activity of the lectin pathway in homozygous (- / -) mouse sera. Mouse serum was added to the confluent-coated wells and the amount of intact C3 attached to the wells was measured. This amount decreased in the deficient serum and was about 21% of normal serum diluted 1/10 (Figure 23B).

Reconstruction of MBL-MASP-sMAP complex in homozygous (- / -) mouse serum

When the recombinant mouse sMAP (rsMAP) or the inactive mouse MASP-2 mutant (MASP-2i) is added to the homozygous (- / -) mouse serum, both recombinants can bind MBL (Fig. 24A, lane 3 and 4). When rsMAP and MASP-2i are incubated with serum at the same time (Fig. 24A, lane 5), both recombinants are detected in the MBL-MASP-sMAP complex. However, the amount of sMAP bound to the complex was lower than that of rsMAP alone. The competitive binding of sMAP and MASP-2 to MBL was then further investigated. A certain amount of rsMAP and various amounts of MASP-2i were added to deficient serum. The binding of rsMAP was reduced in a dose-dependent manner by an increased amount of MASP-2i (Figure 24B). Conversely, the amount of MASP-2i bound to MBL was reduced by the addition of rsMAP (Figure 24C). When rsMAP was added to wild-type sera, the binding of intrinsic sMAP and MASP-2 to MBL decreased in a dose-dependent manner (Figure 24D).

Reconstitution of C4 adherence activity in homozygous (- / -) mouse serum

Reconstitution experiments of C4 adherence on mannan-coated wells were performed using recombinants. When rsMAP was added to deficient serum, the amount of C4 adhered was virtually reduced to a basal level within a dose-dependent manner (Fig. 25A). When rMASP-2 was added to serum, the amount of C4 recovered to 46% of the wild-type sera in the dose-dependent manner, reaching a peak (Fig. 25B). Next, we investigated the effect of sMAP on C4 attachment. When a constant amount of rMASP-2 and various amounts of rsMAP were added to deficient serum, the amount of adhered C4 was reduced by the addition of rsMAP in a dose-dependent manner (Fig. 26A) and rsMAP was added to the wild- (Fig. 26B), which means that sMAP plays a regulatory role in the activation of the lectin pathway.

discussion

We generated sMAP - / - mice through targeted destruction of sMAP-specific exons. Expression levels of MASP-2 were markedly decreased in both mRNA and protein levels in these mice (Figures 21 and 22). Northern blot analysis by MASP-2 probe shows only additional bands in the poly (A) + RNA of sMAP - / - mice, indicating that the normal splicing of the MASP-2 gene is altered by the targeting of the sMAP gene , The expression level of MASP-2 was significantly reduced. As a result, cleavage of C4 by the MBL-MASP complex in the deficient serum was 80% less than that of the normal serum (Fig. 23A). In the reconstitution experiments, C4 cleavage activity was restored by the addition of rMASP-2 but not rsMAP (Fig. 25). The reduction in C4 adherence observed in deficient serum should be caused by MASP-2 deficiency in the MBL-MASP complex (Figure 22B). Thus, it is clear that MASP-2 is essential for activation of C4 by the MBL-MASP complex. However, the addition of rMASP-2 does not restore complete cleavage activity, and C4 attachment reaches its peak. Most of the rMASP-2 is produced by self-activation during the purification procedure as described above (Cseh et al, J Immunol 169: 5735-5743 (2002); Iwaki and Fujita, J Endotoxin Res 11: 47-50 And some of them lose their protease activity. Since the active or inactive MASP-2 does not have a significant effect on their MBL binding (Zundel et al, J Immunol 172: 4342-4350 (2004)), rMASP-2, which lost its protease activity, It is possible to combine and prevent the binding of the active form competitively, resulting in incomplete recovery of C4 attachment. Lt; RTI ID = 0.0 &gt; C3 &lt; / RTI &gt; mitotic activity deficient serum of the lectin pathway (Figure 23B). The decrease in the amount of attached C3 is probably due to a very low level of C3 converting enzyme activity and consists of C4b and C2a fragments produced by MASP-2.

As a homodimer, MASP and sMAP bind and form complexes with MBL or L-picoline through their N-terminal CUB and EGF-like domains (Chen and Wallis, J Biol Chem 276: 25894-25902 Immunol 169: 5735-5743 (2002); Thielens et al, J Immunol 166: 5068-5077 (2001); Zundel et al., J Immunol 172: 4342-4350 (2004)). The crystal structure of the CUBI-EGF-CUB2 portion of sMAP and MASP-2 reveals their homodimeric structures (Feinberg et al., EMBO J 22: 2348-2359 (2003); Gregory et al., J Biol Chem 278: 32157 -32164 (2003)). The collagen-like domain of MBL is involved in the binding of MASP (Wallis and Cheng, J Immunol 163: 4953-4959 (1999); Wallis and Drickamer, J Biol Chem 279: 14065-14073 Mutations reduce the binding of MASP-1 and MASP-2 to the CUBI-EGF-CUB2 portion of MBL (Wallis and Dodd, J Biol Chem 275: 30962-30969 (2000).) MASP-2 and MASP- The binding sites of sMAP and MASP-2 were probably identical (Fig. 3), although the sMAP-binding site of MBL was not identified, , Because the CUBI-EGF domain is identical in sMAP and MASP-2, so it is natural that sMAP and MASP-2 compete with each other and bind to MBL in the reconstitution of MBL-MASP-sMAP complex ( 24). The affinity of sMAP for MBL is lower than that of MASP-2 (Cseh et al, J Immunol 169: 5735-5743 (2002); Thielens et al., J Immunol 166: 5068-5077 (2001)). As shown in Figure 22A, however, the amount of sMAP in the wild-type serum is much greater than that of MASP-2. Thus, sMAP can occupy the MASP-2 / sMAP binding site , Inhibits MASP-2 binding to MBL and consequently reduces C4 cleavage activity of the MBL-MASP complex. The mechanism of sMAP regulation in the lectin pathway has not been investigated. Whether sMAP acts as a regulator after complement activation SMAP is able to prevent the unconscious activation of the MBL-MASP complex before microbial infection or to inhibit hyperactivation of the lectin pathway when activated. There are other potential modulators in the lectin pathway. MASP-3 is also a competitor of MASP-2 upon binding to MBL and down-regulates the C4 and C2 cleavage activities of MASP-2 (Dahl et al, Immunity 15: 127-135 (2001)). The interaction between sMAP and MASP-3 has not been investigated, but it is possible to down-regulate the activation of the lectin pathway by synergism.

In this study, we demonstrated that sMAP and MASP-2 competed to bind MBL, and sMAP possessed the ability to down-regulate the activated lectin pathway by the MBL-MASP complex. It is reasonable that sMAP regulates the lectin pathway of another pathway activated by the picolin-MASP complex. MASP-2 and sMAP also compete for binding to mouse picoline A and down-regulate the C4 cleavage activity of the picoline A-MASP complex (Y Endo et al, in the product). Studies on mouse MBL null mice have recently been reported (Shi et al., J Exp Med 199: 1379-1390 (2004)). MBL null mice do not have C4 cleavage activity in the MBL lectin pathway and are susceptible to Staphylococcus aureus infection In the present study, sMAP - / - mice deficient in MASP-2 exhibit a decrease in C3 mitogenicity along with C4 cleavage activity in the lectin pathway. Due to their impaired opsonization activity, sMAP-deficient mice Is susceptible to bacterial infections. Further investigation of sMAP-deficient mice will clarify the function of the lectin pathway in protecting against infectious diseases.

Another important finding is the addition of rsMAP to normal serum leads to a decrease in C4 activation (Fig. 26B). The lectin pathway has been shown to regulate inflammation and tissue damage in a number of organs (De Vries et al, Am J Pathol 165: 1677-1688 (2004); Fiane et al, Circulation 108: 849-856 (2003); Jordan et al., Circulation 104: 1413-1418 (2001); Walsh et al., J Immunol 175: 541-546 (2005)). In MBL-deficient patients treated with thoracic abdominal aortic aneurysm, complement was not activated and levels of proinflammatory markers decreased postoperatively (Fiane et al, Circulation 108: 849-856 (2003)). Accumulated evidence has demonstrated the potential pathophysiological role of MBL in various vascular ischemic and reperfusion symptoms. Thus, specific blockade of MBL or inhibition of the lectin complement pathway represents a therapeutic related strategy to prevent ischemia / perfusion-induced damage. Therefore, sMAP can be one of the inhibitor candidates because they act as an attenuator of lectin pathway activation.

Example 31

This example demonstrates that MASP-2 plays a role in the C4 bypass activation of C3.

Background / Rationale : Recently, it has been shown that inhibition of alternative pathways protects the kidney from ischemic acute dysfunction (Thurman et al., J. Immunol 170: 1517-1523 (2003)). The data herein indicate that the lectin pathway directs alternative pathway-activation, which, in turn, synergistically amplifies complement activation. We hypothesize that transient suppression of the lectin pathway affects alternative pathway-activation, thus limiting long-term outcome of organ transplantation by limiting complement-mediated graft injury and inflammation and alleviating unwanted induction of acquired immune responses to grafts , And decreased the secondary graft rejection through the acquired immune system. This is supported by recent clinical data showing partial impairment of the lectin pathway by congenital MBL deficiency (occurring in approximately 30% of the population) and is related to the human survival of increased renal allograft transplantation (Berger, Am J Transplant 5: 1361-1366 (2005)).

The involvement of complement components C3 and C4 in ischemia-reperfusion (I / R) injury was well established in models of transient intestinal and muscle ischemia using gene targeting mouse strains (Weiser et al., J Exp Med 183: 2342- 2348 (1996); Williams et al. J Appl Physiol 86: 938-42, (1999)). C3 is known to play a significant role in renal VR injury and secondary graft rejection (Zhou et al., J Chn Invest 105: 1363-1371 (2000); Pratt et al., Nat Med 8: 582-587 2002); Farrar, et al., Am J. Pathol 164: 133-141 (2004)). It is surprising that the phenotype of C4 deficiency is not observed in the mouse kidney allograft rejection model (Lin, 2005 In Press). Subsequent analysis of serum and plasma of such C4-deficient mice, however, means that these mice retain residual functional activity indicative of LP-dependent cleavage of C3 and further complement down-activation (Figure 27C).

The presence of functional C4-bypass (C2-bypass) is a phenomenon described by many researchers (such as that is not fully characterized) (Miller et al., Proc Natl Acad Sci 72: 418-22 (1975); Knutzen Steuer (1989); Wagner et al., J Immunol 163: 3549-3558 (1999), which suggests that alternative pathway-independent C3-conversion in C4 (and C2) .

METHODS : The effect of lectin pathway and classical pathway on C3 attachment. Diluted mouse plasma (EGTA / Mg2 +, an anticoagulant), and, 4.0 mM barbiturates, 145 mM NaCl, 2.0 mM CaCl 2, 1.0 mM MgCl 2, was re-calcium screen to pH 7.4, then met (shown in Figs. 27A and 27C (As shown in Figure 27B) or microtiter plates coated with zymosan (as shown in Figure 27B) and incubated at 37 [deg.] C for 90 minutes. Plates were washed three times with 10 mM Tris-Cl, 140 mM NaCl, 5.OmM CaCl ₂ , 0.05% Tween 20, pH 7.4 and then C3b attachment was measured using anti-mouse C3c antibody.

Results: The results shown in Figure 27 AC are representative of three independent experiments. When using the same serum in wells coated with immunoglobulin complex instead of mannan or zymosan, C3b attachment and factor B cleavage was observed in MASP-2 (- / -) sera forming WT (+ / +) mouse serum and pool But not in C1q deficient serum (data not shown). This means that alternative pathway activation can be restored in MASP-2 - / - sera when the initiation C3b is provided via CP activity. Figure 27C shows the surprising fact that C3 can be effectively activated in a lectin path-dependent manner in C4 (- / -) deficient plasma. This "C4 bypass" is blocked by inhibition of lectin pathway-activation through pre-incubation of plasma with solubilized mannose or mannose.

C3b attachment to mannan and gemic acid is severely impaired in MASP-2 (- / -) deficient mice and allows all three pathways under experimental conditions according to previous articles on alternative pathway activation. As shown in FIGS. 27A-C, MASP-2 (- / -) deficient mouse plasma does not activate C4 through the lectin pathway nor destroy C3 through the lectin pathway or alternative pathway. We therefore hypothesized that MASP-2 is required for this C4-bypass. Further advances in identifying components that appear to be involved in lectin pathway-dependent C4-bypass are recently reported by Professor Teizo Fujita. Plasma of C4 deficient mice crossed with Fujita MASP- 1/3 deficient mouse strains lost the ability of C3 degradation by the lectin pathway of C4 deficient plasma. This was restored by the addition of recombinant MASP-1 to combined C4 and MASP-1/3 deficient plasma (Takahashi, MoI Immunol 43: 153 (2006) (Recombinant MASP-1 degrades C2, but not C3; Rossi et al, J Biol Chem 276: 40880-7 (2001); Chen et al, J Biol Chem 279 : 26058-65 (2004). We observed that MASP-2 was required for this bypass.

Although more functional and quantitative variables and histology are needed to consolidate this pilot study, their preliminary results indicate that complement activation by the lectin pathway contributes significantly to the pathophysiology of renal I / R injury and MASP-2- / - strongly supports the hypothesis that mice exhibit faster renal function recovery.

While a preferred embodiment of the invention has been illustrated and described, it should be understood that various changes may be made without departing from the spirit and scope of the invention.

                         SEQUENCE LISTING <120> METHODS FOR TREATING CONDITIONS ASSOCIATED WITH MASP-2 DEPENDENT        COMPLEMENT ACTIVATION <130> OMER-1-29200 &Lt; 150 > US 11 / 645,359 <151> 2006-12-22 &Lt; 150 > US 60 / 788,876 <151> 2006-04-03 &Lt; 150 > US 60 / 578,847 <151> 2004-06-10 <150> US 11 / 150,883 <151> 2005-06-09 <160> 65 <170> PatentIn version 3.2 <210> 1 <211> 725 <212> DNA <213> Homo sapien <220> <221> CDS &Lt; 222 > (27) .. (584) <400> 1 ggccaggcca gctggacggg cacacc atg agg ctg ctg acc ctc ctg ggc ctt 53                              Met Arg Leu Leu Thr Leu Leu Gly Leu                              1 5 ctg tgt ggc tcg gtg gcc acc ccc ttg ggc ccg aag tgg cct gaa cct 101 Leu Cys Gly Ser Val Ala Thr Pro Leu Gly Pro Lys Trp Pro Glu Pro 10 15 20 25 gtg ttc ggg cgc ctg gca tcc ccc ggc ttt cca ggg gag tat gcc aat 149 Val Phe Gly Arg Leu Ala Ser Pro Gly Phe Pro Gly Glu Tyr Ala Asn                 30 35 40 gac cag gag cgg cgc tgg acc ctg act gca ccc ccc ggc tac cgc ctg 197 Asp Gln Glu Arg Arg Trp Thr Leu Thr Ala Pro Pro Gly Tyr Arg Leu             45 50 55 cgc ctc tac ttc acc cac ttc gac ctg gag ctc tcc cac ctc tgc gag 245 Arg Leu Tyr Phe Thr His Phe Asp Leu Glu Leu Ser His Leu Cys Glu         60 65 70 tac gac ttc gtc aag ctg agc tcg ggg gcc aag gtg ctg gcc acg ctg 293 Tyr Asp Phe Val Lys Leu Ser Ser Gly Ala Lys Val Leu Ala Thr Leu     75 80 85 tgc ggg cag gag agc aca gac acg gag cgg gcc cct ggc aag gac act 341 Cys Gly Gln Glu Ser Thr Asp Thr Glu Arg Ala Pro Gly Lys Asp Thr 90 95 100 105 ttc tac tcg ctg ggc tcc agc ctg gac att acc ttc cgc tcc gac tac 389 Phe Tyr Ser Leu Gly Ser Ser Leu Asp Ile Thr Phe Arg Ser Asp Tyr                 110 115 120 tcc aac gag aag ccg ttc acg ggg ttc gag gcc ttc tat gca gcc gag 437 Ser Asn Glu Lys Pro Phe Thr Gly Phe Glu Ala Phe Tyr Ala Ala Glu             125 130 135 gac att gac gag tgc cag gtg gcc ccg gga gag gcg ccc acc tgc gac 485 Asp Ile Asp Glu Cys Gln Val Ala Pro Gly Glu Ala Pro Thr Cys Asp         140 145 150 cac cac tgc cac aac cac ctg ggc ggt ttc tac tgc tcc tgc cgc gca 533 His His Cys His Asn His Leu Gly Gly Phe Tyr Cys Ser Cys Arg Ala     155 160 165 ggc tac gtc ctg cac cgt aac aag cgc acc tgc tca gag cag agc ctc 581 Gly Tyr Val Leu His Arg Asn Lys Arg Thr Cys Ser Glu Gln Ser Leu 170 175 180 185 tag cctcccctgg agctccggcc tgcccagcag gtcagaagcc agagccagcc 634 tgctggcctc agctccgggt tgggctgaga tggctgtgcc ccaactccca ttcacccacc 694 atggacccaa taataaacct ggccccaccc c 725 <210> 2 <211> 185 <212> PRT <213> Homo sapien <400> 2 Met Arg Leu Leu Thr Leu Leu Gly Leu Leu Cys Gly Ser Val Ala Thr 1 5 10 15 Pro Leu Gly Pro Lys Trp Pro Glu Pro Val Phe Gly Arg Leu Ala Ser             20 25 30 Pro Gly Phe Pro Gly Glu Tyr Ala Asn Asp Gln Glu Arg Arg Trp Thr         35 40 45 Leu Thr Ala Pro Pro Gly Tyr Arg Leu Arg Leu Tyr Phe Thr His Phe     50 55 60 Asp Leu Glu Leu Ser His Leu Cys Glu Tyr Asp Phe Val Lys Leu Ser 65 70 75 80 Ser Gly Ala Lys Val Leu Ala Thr Leu Cys Gly Gln Glu Ser Thr Asp                 85 90 95 Thr Glu Arg Ala Pro Gly Lys Asp Thr Phe Tyr Ser Leu Gly Ser Ser             100 105 110 Leu Asp Ile Thr Phe Arg Ser Asp Tyr Ser Asn Glu Lys Pro Phe Thr         115 120 125 Gly Phe Glu Ala Phe Tyr Ala Ala Glu Asp Ile Asp Glu Cys Gln Val     130 135 140 Ala Pro Gly Glu Ala Pro Thr Cys Asp His His Cys His Asn His Leu 145 150 155 160 Gly Gly Phe Tyr Cys Ser Cys Arg Ala Gly Tyr Val Leu His Arg Asn                 165 170 175 Lys Arg Thr Cys Ser Glu Gln Ser Leu             180 185 <210> 3 <211> 170 <212> PRT <213> Homo sapien <400> 3 Thr Pro Leu Gly Pro Lys Trp Pro Glu Pro Val Phe Gly Arg Leu Ala 1 5 10 15 Ser Pro Gly Phe Pro Gly Glu Tyr Ala Asn Asp Gln Glu Arg Arg Trp             20 25 30 Thr Leu Thr Ala Pro Pro Gly Tyr Arg Leu Arg Leu Tyr Phe Thr His         35 40 45 Phe Asp Leu Glu Leu Ser His Leu Cys Glu Tyr Asp Phe Val Lys Leu     50 55 60 Ser Ser Gly Ala Lys Val Leu Ala Thr Leu Cys Gly Gln Glu Ser Thr 65 70 75 80 Asp Thr Glu Arg Ala Pro Gly Lys Asp Thr Phe Tyr Ser Leu Gly Ser                 85 90 95 Ser Leu Asp Ile Thr Phe Arg Ser Asp Tyr Ser Asn Glu Lys Pro Phe             100 105 110 Thr Gly Phe Glu Ala Phe Tyr Ala Ala Glu Asp Ile Asp Glu Cys Gln         115 120 125 Val Ala Pro Gly Glu Ala Pro Thr Cys Asp His His Cys His Asn His     130 135 140 Leu Gly Gly Phe Tyr Cys Ser Cys Arg Ala Gly Tyr Val Leu His Arg 145 150 155 160 Asn Lys Arg Thr Cys Ser Glu Gln Ser Leu                 165 170 <210> 4 <211> 2460 <212> DNA <213> Homo sapien <220> <221> CDS (22). (2082) <400> 4 ggccagctgg acgggcacac c atg agg ctg ctg acc ctc ctg ggc ctt ctg 51                         Met Arg Leu Leu Thr Leu Leu Gly Leu Leu                         1 5 10 tgt ggc tcg gtg gcc acc ccc ttg ggc ccg aag tgg cct gaa cct gtg 99 Cys Gly Ser Val Ala Thr Pro Leu Gly Pro Lys Trp Pro Glu Pro Val                 15 20 25 ttc ggg cgc ctg gcc tcc ccc ggc ttt cca ggg gag tat gcc aat gac 147 Phe Gly Arg Leu Ala Ser Pro Gly Phe Pro Gly Glu Tyr Ala Asn Asp             30 35 40 cag gag cgg cgc tgg acc ctg act gca ccc ccc ggc tac cgc ctg cgc 195 Gln Glu Arg Arg Trp Thr Leu Thr Ala Pro Pro Gly Tyr Arg Leu Arg         45 50 55 ctc tac ttc acc cac ttc gac ctg gag ctc tcc cac ctc tgc gag tac 243 Leu Tyr Phe Thr His Phe Asp Leu Glu Leu Ser His Leu Cys Glu Tyr     60 65 70 gac ttc gtc aag ctg agc tcg ggg gcc aag gtg ctg gcc acg ctg tgc 291 Asp Phe Val Lys Leu Ser Ser Gly Ala Lys Val Leu Ala Thr Leu Cys 75 80 85 90 ggg cag gag agc aca gac acg gag cgg gcc cct ggc aag gac act ttc 339 Gly Gln Glu Ser Thr Asp Thr Glu Arg Ala Pro Gly Lys Asp Thr Phe                 95 100 105 tac tcg ctg ggc tcc agc ctg gac att acc ttc cgc tcc gac tac tcc 387 Tyr Ser Leu Gly Ser Ser Leu Asp Ile Thr Phe Arg Ser Asp Tyr Ser             110 115 120 aac gag aag ccg ttc acg ggg ttc gag gcc ttc tat gca gcc gag gac 435 Asn Glu Lys Pro Phe Thr Gly Phe Glu Ala Phe Tyr Ala Ala Glu Asp         125 130 135 att gac gag tgc cag gtg gcc ccg gga gag gcg ccc acc tgc gac cac 483 Ile Asp Glu Cys Gln Val Ala Pro Gly Glu Ala Pro Thr Cys Asp His     140 145 150 cac tgc cac aac cac ctg ggc ggt ttc tac tgc tcc tgc cgc gca ggc 531 His Cys His Asn His Leu Gly Gly Phe Tyr Cys Ser Cys Arg Ala Gly 155 160 165 170 tac gtc ctg cac cgt aac aag cgc acc tgc tca gcc ctg tgc tcc ggc 579 Tyr Val Leu His Arg Asn Lys Arg Thr Cys Ser Ala Leu Cys Ser Gly                 175 180 185 cag gtc ttc acc cag agg tct ggg gag ctc agc agc cct gaa tac cca 627 Gln Val Phe Thr Gln Arg Ser Gly Glu Leu Ser Ser Pro Glu Tyr Pro             190 195 200 cgg ccg tat ccc aaa ctc tcc agt tgc act tac agc atc agc ctg gag 675 Arg Pro Tyr Pro Lys Leu Ser Ser Cys Thr Tyr Ser Ile Ser Leu Glu         205 210 215 gag ggg ttc agt gtc att ctg gac ttt gtg gag tcc ttc gat gtg gag 723 Glu Gly Phe Ser Val Ile Leu Asp Phe Val Glu Ser Phe Asp Val Glu     220 225 230 aca cac cct gaa acc ctg tgt ccc tac gac ttt ctc aag att caa aca 771 Thr His Pro Glu Thr Leu Cys Pro Tyr Asp Phe Leu Lys Ile Gln Thr 235 240 245 250 gac aga gaa gaa cat ggc cca ttc tgt ggg aag aca ttg ccc cac agg 819 Asp Arg Glu Glu His Gly Pro Phe Cys Gly Lys Thr Leu Pro His Arg                 255 260 265 att gaa aca aaa agc aac acg gtg acc acac ttt gtc aca gat gaa 867 Ile Glu Thr Lys Ser Asn Thr Val Thr Ile Thr Phe Val Thr Asp Glu             270 275 280 tca gga gac cac aca ggc tgg aag atc cac tac acg acc aca gcg cag 915 Ser Gly Asp His Thr Gly Trp Lys Ile His Tyr Thr Ser Thr Ala Gln         285 290 295 cct tgc cct tat ccg atg gcg cca cct aat ggc cac gtt tca cct gtg 963 Pro Cys Pro Tyr Pro Met Ala Pro Pro Asn Gly His Val Val Ser Pro Val     300 305 310 caa gcc aaa tac atc ctg aaa gac agc ttc tcc atc ttt tgc gag act 1011 Gln Ala Lys Tyr Ile Leu Lys Asp Ser Phe Ser Ile Phe Cys Glu Thr 315 320 325 330 ggc tat gag ctt ctg caa ggt cac ttg ccc ctg aaa tcc ttt act gca 1059 Gly Tyr Glu Leu Leu Gln Gly His Leu Pro Leu Lys Ser Phe Thr Ala                 335 340 345 gtt tgt cag aaa gat gga tct tgg gac cgg cca atg ccc gcg tgc agc 1107 Val Cys Gln Lys Asp Gly Ser Trp Asp Arg Pro Met Pro Ala Cys Ser             350 355 360 att gtt gac tgt ggc cct cct gat gat cta ccc agt ggc cga gtg gag 1155 Ile Val Asp Cys Gly Pro Pro Asp Asp Leu Pro Ser Gly Arg Val Glu         365 370 375 tac atc aca ggt cct gga gtg acc acc tac aaa gct gtg att cag tac 1203 Tyr Ile Thr Gly Pro Gly Val Thr Thr Tyr Lys Ala Val Ile Gln Tyr     380 385 390 agc tgt gaa gag acc ttc tac aca atg aaa gtg aat gat ggt aaa tat 1251 Ser Cys Glu Glu Thr Phe Tyr Thr Met Lys Val Asn Asp Gly Lys Tyr 395 400 405 410 gtg tgt gag gct gat gga ttc tgg acg agc tcc aaa gga gaa aaa tca 1299 Val Cys Glu Ala Asp Gly Phe Trp Thr Ser Ser Lys Gly Glu Lys Ser                 415 420 425 ctc cca gtc tgt gag cct gtt tgt gga cta tca gcc cgc aca aca gga 1347 Leu Pro Val Cys Glu Pro Val Cys Gly Leu Ser Ala Arg Thr Thr Gly             430 435 440 ggg cgt ata tat gga ggg caa aag gca aaa cct ggt gat ttt cct tgg 1395 Gly Arg Ile Tyr Gly Gly Gln Lys Ala Lys Pro Gly Asp Phe Pro Trp         445 450 455 caa gtc ctg ata tta ggt gga acc aca gca gca ggt gca ctt tta tat 1443 Gln Val Leu Ile Leu Gly Gly Thr Thr Ala Ala Gly Ala Leu Leu Tyr     460 465 470 gac aac tgg gtc cta aca gct gct cat gcc gtc tat gag caa aaa cat 1491 Asp Asn Trp Val Leu Thr Ala Ala His Ala Val Tyr Glu Gln Lys His 475 480 485 490 gat gca tcc gcc ctg gac att cga atg ggc acc ctg aaa aga cta tca 1539 Asp Ala Ser Ala Leu Asp Ile Arg Met Gly Thr Leu Lys Arg Leu Ser                 495 500 505 cct cat tat aca caa gcc tgg tct gaa gct gtt ttt ata cat gaa ggt 1587 Pro His Tyr Thr Gln Ala Trp Ser Glu Ala Val Phe Ile His Glu Gly             510 515 520 tat act cat gat gct ggc ttt gac aat gac ata gca ctg att aaa ttg 1635 Tyr Thr His Asp Ala Gly Phe Asp Asn Asp Ile Ala Leu Ile Lys Leu         525 530 535 aat aac aaa gtt gta atc aat agc aac atc acg cct att tgt ctg cca 1683 Asn Asn Lys Val Ile Asn Ser Asn Ile Thr Pro Ile Cys Leu Pro     540 545 550 aga aaa gaa gct gaa tcc ttt atg agg aca gat gac att gga act gca 1731 Arg Lys Glu Ala Glu Ser Phe Met Arg Thr Asp Asp Ile Gly Thr Ala 555 560 565 570 tct gga tgg gga tta acc caa agg ggt ttt ctt gct aga aat cta atg 1779 Ser Gly Trp Gly Leu Thr Gln Arg Gly Phe Leu Ala Arg Asn Leu Met                 575 580 585 taste gtc gac ata ccg att gtt gac cat caa aaa tgt act gct gca tat 1827 Tyr Val Asp Ile Pro Ile Val Asp His Gln Lys Cys Thr Ala Ala Tyr             590 595 600 gaa aag cca ccc tat cca agg gga agt gta act gct aac atg ctt tgt 1875 Glu Lys Pro Pro Tyr Pro Arg Gly Ser Val Thr Ala Asn Met Leu Cys         605 610 615 gct ggc tta gaa agt ggg ggc aag gac agc tgc aga ggt gac agc gga 1923 Ala Gly Leu Glu Ser Gly Gly Lys Asp Ser Cys Arg Gly Asp Ser Gly     620 625 630 ggg gca ctg gtg ttt cta gat agt gaa aca gag agg tgg ttt gtg gga 1971 Gly Ala Leu Val Phe Leu Asp Ser Glu Thr Glu Arg Trp Phe Val Gly 635 640 645 650 gga ata gtg tcc tgg ggt tcc atg aat tgt ggg gaa gca ggt cag tat 2019 Gly Ile Val Ser Trp Gly Ser Met Asn Cys Gly Glu Ala Gly Gln Tyr                 655 660 665 gga gtc tac aca aaa gtt att aac tat att ccc tgg atc gag aac ata 2067 Gly Val Tyr Thr Lys Val Ile Asn Tyr Ile Pro Trp Ile Glu Asn Ile             670 675 680 att agt gat ttt taa cttgcgtgtc tgcagtcaag gattcttcat ttttagaaat 2122 Ile Ser Asp Phe         685 gcctgtgaag accttggcag cgacgtggct cgagaagcat tcatcattac tgtggacatg 2182 gcagttgttg ctccacccaa aaaaacagac tccaggtgag gctgctgtca tttctccact 2242 tgccagttta attccagcct tacccattga ctcaagggga cataaaccac gagagtgaca 2302 gtcatctttg cccacccagt gtaatgtcac tgctcaaatt acatttcatt accttaaaaa 2362 gccagtctct tttcatactg gctgttggca tttctgtaaa ctgcctgtcc atgctctttg 2422 tttttaaact tgttcttatt gaaaaaaaaa aaaaaaaa 2460 <210> 5 <211> 686 <212> PRT <213> Homo sapien <400> 5 Met Arg Leu Leu Thr Leu Leu Gly Leu Leu Cys Gly Ser Val Ala Thr 1 5 10 15 Pro Leu Gly Pro Lys Trp Pro Glu Pro Val Phe Gly Arg Leu Ala Ser             20 25 30 Pro Gly Phe Pro Gly Glu Tyr Ala Asn Asp Gln Glu Arg Arg Trp Thr         35 40 45 Leu Thr Ala Pro Pro Gly Tyr Arg Leu Arg Leu Tyr Phe Thr His Phe     50 55 60 Asp Leu Glu Leu Ser His Leu Cys Glu Tyr Asp Phe Val Lys Leu Ser 65 70 75 80 Ser Gly Ala Lys Val Leu Ala Thr Leu Cys Gly Gln Glu Ser Thr Asp                 85 90 95 Thr Glu Arg Ala Pro Gly Lys Asp Thr Phe Tyr Ser Leu Gly Ser Ser             100 105 110 Leu Asp Ile Thr Phe Arg Ser Asp Tyr Ser Asn Glu Lys Pro Phe Thr         115 120 125 Gly Phe Glu Ala Phe Tyr Ala Ala Glu Asp Ile Asp Glu Cys Gln Val     130 135 140 Ala Pro Gly Glu Ala Pro Thr Cys Asp His His Cys His Asn His Leu 145 150 155 160 Gly Gly Phe Tyr Cys Ser Cys Arg Ala Gly Tyr Val Leu His Arg Asn                 165 170 175 Lys Arg Thr Cys Ser Ala Leu Cys Ser Gly Gln Val Phe Thr Gln Arg             180 185 190 Ser Gly Glu Leu Ser Ser Pro Glu Tyr Pro Arg Pro Tyr Pro Lys Leu         195 200 205 Ser Ser Cys Thr Tyr Ser Ile Ser Leu Glu Glu Gly Phe Ser Val Ile     210 215 220 Leu Asp Phe Val Glu Ser Phe Asp Val Glu Thr His Pro Glu Thr Leu 225 230 235 240 Cys Pro Tyr Asp Phe Leu Lys Ile Gln Thr Asp Arg Glu Glu His Gly                 245 250 255 Pro Phe Cys Gly Lys Thr Leu Pro His Arg Ile Glu Thr Lys Ser Asn             260 265 270 Thr Val Thr Ile Thr Phe Val Thr Asp Glu Ser Gly Asp His Thr Gly         275 280 285 Trp Lys Ile His Tyr Thr Ser Thr Ala Gln Pro Cys Pro Tyr Pro Met     290 295 300 Ala Pro Pro Asn Gly His Val Ser Pro Val Gln Ala Lys Tyr Ile Leu 305 310 315 320 Lys Asp Ser Phe Ser Ile Phe Cys Glu Thr Gly Tyr Glu Leu Leu Gln                 325 330 335 Gly His Leu Pro Leu Lys Ser Phe Thr Ala Val Cys Gln Lys Asp Gly             340 345 350 Ser Trp Asp Arg Pro Met Pro Ala Cys Ser Ile Val Asp Cys Gly Pro         355 360 365 Pro Asp Leu Pro Ser Gly Arg Val Glu Tyr Ile Thr Gly Pro Gly     370 375 380 Val Thr Thr Tyr Lys Ala Val Ile Gln Tyr Ser Cys Glu Glu Thr Phe 385 390 395 400 Tyr Thr Met Lys Val Asn Asp Gly Lys Tyr Val Cys Glu Ala Asp Gly                 405 410 415 Phe Trp Thr Ser Ser Lys Gly Glu Lys Ser Leu Pro Val Cys Glu Pro             420 425 430 Val Cys Gly Leu Ser Ala Arg Thr Thr Gly Gly Arg Ile Tyr Gly Gly         435 440 445 Gln Lys Ala Lys Pro Gly Asp Phe Pro Trp Gln Val Leu Ile Leu Gly     450 455 460 Gly Thr Thr Ala Ala Gly Ala Leu Leu Tyr Asp Asn Trp Val Leu Thr 465 470 475 480 Ala Ala His Ala Val Tyr Glu Gln Lys His Asp Ala Ser Ala Leu Asp                 485 490 495 Ile Arg Met Gly Thr Leu Lys Arg Leu Ser Pro His Tyr Thr Gln Ala             500 505 510 Trp Ser Glu Ala Val Phe Ile His Glu Gly Tyr Thr His Asp Ala Gly         515 520 525 Phe Asp Asn Asp Ile Ala Leu Ile Lys Leu Asn Asn Lys Val Val Ile     530 535 540 Asn Ser Asn Ile Thr Pro Ile Cys Leu Pro Arg Lys Glu Ala Glu Ser 545 550 555 560 Phe Met Arg Thr Asp Asp Ile Gly Thr Ala Ser Gly Trp Gly Leu Thr                 565 570 575 Gln Arg Gly Phe Leu Ala Arg Asn Leu Met Tyr Val Asp Ile Pro Ile             580 585 590 Val Asp His Gln Lys Cys Thr Ala Ala Tyr Glu Lys Pro Pro Tyr Pro         595 600 605 Arg Gly Ser Val Thr Ala Asn Met Leu Cys Ala Gly Leu Glu Ser Gly     610 615 620 Gly Lys Asp Ser Cys Arg Gly Asp Ser Gly Gly Ala Leu Val Phe Leu 625 630 635 640 Asp Ser Glu Thr Glu Arg Trp Phe Val Gly Gly Ile Val Ser Trp Gly                 645 650 655 Ser Met Asn Cys Gly Glu Ala Gly Gln Tyr Gly Val Tyr Thr Lys Val             660 665 670 Ile Asn Tyr Ile Pro Trp Ile Glu Asn Ile Ile Ser Asp Phe         675 680 685 <210> 6 <211> 671 <212> PRT <213> Homo sapien <400> 6 Thr Pro Leu Gly Pro Lys Trp Pro Glu Pro Val Phe Gly Arg Leu Ala 1 5 10 15 Ser Pro Gly Phe Pro Gly Glu Tyr Ala Asn Asp Gln Glu Arg Arg Trp             20 25 30 Thr Leu Thr Ala Pro Pro Gly Tyr Arg Leu Arg Leu Tyr Phe Thr His         35 40 45 Phe Asp Leu Glu Leu Ser His Leu Cys Glu Tyr Asp Phe Val Lys Leu     50 55 60 Ser Ser Gly Ala Lys Val Leu Ala Thr Leu Cys Gly Gln Glu Ser Thr 65 70 75 80 Asp Thr Glu Arg Ala Pro Gly Lys Asp Thr Phe Tyr Ser Leu Gly Ser                 85 90 95 Ser Leu Asp Ile Thr Phe Arg Ser Asp Tyr Ser Asn Glu Lys Pro Phe             100 105 110 Thr Gly Phe Glu Ala Phe Tyr Ala Ala Glu Asp Ile Asp Glu Cys Gln         115 120 125 Val Ala Pro Gly Glu Ala Pro Thr Cys Asp His His Cys His Asn His     130 135 140 Leu Gly Gly Phe Tyr Cys Ser Cys Arg Ala Gly Tyr Val Leu His Arg 145 150 155 160 Asn Lys Arg Thr Cys Ser Ala Leu Cys Ser Gly Gln Val Phe Thr Gln                 165 170 175 Arg Ser Gly Glu Leu Ser Ser Pro Glu Tyr Pro Arg Pro Tyr Pro Lys             180 185 190 Leu Ser Ser Cys Thr Tyr Ser Ile Ser Leu Glu Glu Gly Phe Ser Val         195 200 205 Ile Leu Asp Phe Val Glu Ser Phe Asp Val Glu Thr His Pro Glu Thr     210 215 220 Leu Cys Pro Tyr Asp Phe Leu Lys Ile Gln Thr Asp Arg Glu Glu His 225 230 235 240 Gly Pro Phe Cys Gly Lys Thr Leu Pro His Arg Ile Glu Thr Lys Ser                 245 250 255 Asn Thr Val Thr Ile Thr Phe Val Thr Asp Glu Ser Gly Asp His Thr             260 265 270 Gly Trp Lys Ile His Tyr Thr Ser Thr Ala Gln Pro Cys Pro Tyr Pro         275 280 285 Met Ala Pro Pro Asn Gly His Val Ser Pro Val Gln Ala Lys Tyr Ile     290 295 300 Leu Lys Asp Ser Phe Ser Ile Phe Cys Glu Thr Gly Tyr Glu Leu Leu 305 310 315 320 Gln Gly His Leu Pro Leu Lys Ser Phe Thr Ala Val Cys Gln Lys Asp                 325 330 335 Gly Ser Trp Asp Arg Pro Met Pro Ala Cys Ser Ile Val Asp Cys Gly             340 345 350 Pro Pro Asp Leu Pro Ser Gly Arg Val Glu Tyr Ile Thr Gly Pro         355 360 365 Gly Val Thr Thr Tyr Lys Ala Val Ile Gln Tyr Ser Cys Glu Glu Thr     370 375 380 Phe Tyr Thr Met Lys Val Asn Asp Gly Lys Tyr Val Cys Glu Ala Asp 385 390 395 400 Gly Phe Trp Thr Ser Ser Lys Gly Glu Lys Ser Leu Pro Val Cys Glu                 405 410 415 Pro Val Cys Gly Leu Ser Ala Arg Thr Thr Gly Gly Arg Ile Tyr Gly             420 425 430 Gly Gln Lys Ala Lys Pro Gly Asp Phe Pro Trp Gln Val Leu Ile Leu         435 440 445 Gly Gly Thr Thr Ala Gly Ala Leu Leu Tyr Asp Asn Trp Val Leu     450 455 460 Thr Ala Ala His Ala Val Tyr Glu Gln Lys His Asp Ala Ser Ala Leu 465 470 475 480 Asp Ile Arg Met Gly Thr Leu Lys Arg Leu Ser Pro His Tyr Thr Gln                 485 490 495 Ala Trp Ser Glu Ala Val Phe Ile His Glu Gly Tyr Thr His Asp Ala             500 505 510 Gly Phe Asp Asn Asp Ile Ala Leu Ile Lys Leu Asn Asn Lys Val Val         515 520 525 Ile Asn Ser Asn Ile Thr Pro Ile Cys Leu Pro Arg Lys Glu Ala Glu     530 535 540 Ser Phe Met Arg Thr Asp Asp Ile Gly Thr Ala Ser Gly Trp Gly Leu 545 550 555 560 Thr Gln Arg Gly Phe Leu Ala Arg Asn Leu Met Tyr Val Asp Ile Pro                 565 570 575 Ile Val Asp His Gln Lys Cys Thr Ala Ala Tyr Glu Lys Pro Pro Tyr             580 585 590 Pro Arg Gly Ser Val Thr Ala Asn Met Leu Cys Ala Gly Leu Glu Ser         595 600 605 Gly Gly Lys Asp Ser Cys Arg Gly Asp Ser Gly Gly Ala Leu Val Phe     610 615 620 Leu Asp Ser Glu Thr Glu Arg Trp Phe Val Gly Gly Ile Val Ser Trp 625 630 635 640 Gly Ser Met Asn Cys Gly Glu Ala Gly Gln Tyr Gly Val Tyr Thr Lys                 645 650 655 Val Ile Asn Tyr Ile Pro Trp Ile Glu Asn Ile Ile Ser Asp Phe             660 665 670 <210> 7 <211> 4900 <212> DNA <213> Homo sapien <400> 7 cctgtcctgc ctgcctggaa ctctgagcag gctggagtca tggagtcgat tcccagaatc 60 ccagagtcag ggaggctggg ggcaggggca ggtcactgga caaacagatc aaaggtgaga 120 ccagcgtagg actgcagacc aggccaggcc agctggacgg gcacaccatg aggtaggtgg 180 gcgccacagc ctccctgcag ggtgtggggt gggagcacag gcctgggcct caccgcccct 240 gccctgccca taggctgctg accctcctgg gccttctgtg tggctcggtg gccaccccct 300 taggcccgaa gtggcctgaa cctgtgttcg ggcgcctggc atcccccggc tttccagggg 360 agtatgccaa tgaccaggag cggcgctgga ccctgactgc accccccggc taccgcctgc 420 gcctctactt cacccacttc gacctggagc tctcccacct ctgcgagtac gacttcgtca 480 aggtgccgtc agacgggagg gctggggttt ctcagggtcg gggggtcccc aaggagtagc 540 cagggttcag ggacacctgg gagcaggggc caggcttggc caggagggag atcaggcctg 600 ggtcttgcct tcactccctg tgacacctga ccccacagct gagctcgggg gccaaggtgc 660 tggccacgct gtgcgggcag gagagcacag acacggagcg ggcccctggc aaggacactt 720 tctactcgct gggctccagc ctggacatta ccttccgctc cgactactcc aacgagaagc 780 cgttcacggg gttcgaggcc ttctatgcag ccgagggtga gccaagaggg gtcctgcaac 840 atctcagtct gcgcagctgg ctgtgggggt aactctgtct taggccaggc agccctgcct 900 tcagtttccc cacctttccc agggcagggg agaggcctct ggcctgacat catccacaat 960 gcaaagacca aaacagccgt gacctccatt cacatgggct gagtgccaac tctgagccag 1020 ggatctgagg acagcatcgc ctcaagtgac gcagggactg gccgggcgcg gcagctcacg 1080 cctgtaattc cagcactttg ggaggccgag gctggcttga taatttgagg gtcaggagtt 1140 caaggccagc cagggcaaca cggtgaaact ctatctccac taaaactaca aaaattagct 1200 gggcgtggtg gtgcgcacct ggaatcccag ctactaggga ggctgaggca ggagaattgc 1260 ttgaacctgc gaggtggagg ctgcagtgaa cagagattgc accactacac tccacctggg 1320 cgacagacta gactccgtct caaaaaacaa aaaacaaaaa ccacgcaggg ccgagggccc 1380 atttacaagc tgacaaagtg ggccctgcca gcgggagcgc tgcaggatgt ttgattttca 1440 gatcccagtc cctgcagaga ccaactgtgt gacctctggc aagtggctc atttctctgc 1500 tccttagaag ctgctgcaag ggttcagcgc tgtagccccg ccccctgggt ttgattgact 1560 cccctcatta gctgggtgac ctcggccgga cactgaaact cccactggtt taacagaggt 1620 gatgtttgca tctttctccc agcgctgctg ggagcttgca gcgaccctag gcctgtaagg 1680 tgattggccc ggcaccagtc ccgcacccta gacaggacct aggcctcctc tgaggtccac 1740 tctgaggtca tggatctcct gggaggagtc caggctggat cccgcctctt tccctcctga 1800 cggcctgcct ggccctgcct ctcccccaga cattgacgag tgccaggtgg ccccgggaga 1860 ggcgcccacc tgcgaccacc actgccacaa ccacctgggc ggtttctact gctcctgccg 1920 cgcaggctac gtcctgcacc gtaacaagcg cacctgctca ggtgagggag gctgcctggg 1980 ccccaacgca ccctctcctg ggatacccgg ggctcctcag ggccattgct gctctgccca 2040 ggggtgcgga gggcctgggc ctggacactg ggtgcttcta ggccctgctg cctccagctc 2100 cccttctcag ccctgcttcc cctctcagca gccaggctca tcagtgccac cctgccctag 2160 cactgagact aattctaaca tcccactgtg tacctggttc cacctgggct ctgggaaccc 2220 ctcatgtagc cacgggagag tcggggtatc taccctcgtt ccttggactg ggttcctgtt 2280 ccctgcactg ggggacgggc cagtgctctg gggcgtgggc agccccaccc tgtggcgctg 2340 accctgctcc cccgactcgg tttctcctct cggggtctct ccttgcctct ctgatctctc 2400 ttccagagca gagcctctag cctcccctgg agctccggct gcccagcagg tcagaagcca 2460 gagccaggct gctggcctca gctccgggtt gggctgagat gctgtgcccc aactcccatt 2520 cacccaccat ggacccaata ataaacctgg ccccacccca cctgctgccg cgtgtctctg 2580 gggtgggagg gtcgggaggc ggtggggcgc gctcctctct gcctaccctc ctcacagcct 2640 catgaacccc aggtctgtgg gagcctcctc catggggcca cacggtcctt ggcctcaccc 2700 cctgttttga agatggggca ctgaggccgg agaggggtaa ggcctcgctc gagtccaggt 2760 ccccagaggc tgagcccaga gtaatcttga accaccccca ttcagggtct ggcctggagg 2820 agcctgaccc acagaggaga caccctggga gatattcatt gaggggtaat ctggtccccc 2880 gcaaatccag gggtgattcc cactgcccca taggcacagc cacgtggaag aaggcaggca 2940 atgttggggc tcctcacttc ctagaggcct cacaactcaa atgcccccca ctgcagctgg 3000 gggtggggtg gtggtatggg atggggacca agccttcctt gaaggataga gcccagccca 3060 acaccccgcc ccgtggcagc agcatcacgt gttccagcga ggaaggagag caccagactc 3120 agtcatgatc actgttgcct tgaacttcca agaacagccc cagggcaagg gtcaaaacag 3180 gggaaagggg gtgatgagag atccttcttc cggatgttcc tccaggaacc agggggctgg 3240 ctggtcttgg ctgggttcgg gtaggagacc catgatgaat aaacttggga atcactgggg 3300 tggctgtaag ggaatttagg ggagctccga aggggccctt aggctcgagg agatgctcct 3360 ctcttttccc gaattcccag ggacccagga gagtgtccct tcttcctctt cctgtgtgtc 3420 catccacccc cgccccccgc cctggcagag ctggtggaac tcagtgctct agcccctacc 3480 ctggggttgc gactctggct caggacacca ccacgctccc tgggggtgtg agtgagggcc 3540 tgtgcgctcc atcccgagtg ctgcctgttt cagctaaagc ctcaaagcaa gagaaacccc 3600 ctctctaagc ggcccctcag ccatcgggtg ggtcgtttgg tttctgggta ggcctcaggg 3660 gctggccacc tgcagggccc agcccaaccc agggatgcag atgtcccagc cacatccctg 3720 tcccagtttc ctgctcccca aggcatccac cctgctgttg gtgcgagggc tgatagaggg 3780 cacgccaagt cactcccctg cccttccctc cttccagccc tgtgctccgg ccaggtcttc 3840 acccagaggt ctggggagct cagcagccct gaatacccac ggccgtatcc caaactctcc 3900 agttgcactt acagcatcag cctggaggag gggttcagtg tcattctgga ctttgtggag 3960 tccttcgatg tggagacaca ccctgaaacc ctgtgtccct acgactttct caaggtctgg 4020 ctcctgggcc cctcatcttg tcccagatcc tcccccttca gcccagctgc accccctact 4080 tcctgcagca tggcccccac cacgttcccg tcaccctcgg tgaccccacc tcttcaggtg 4140 ctctatggag gtcaaggctg gggcttcgag tacaagtgtg ggaggcagag tggggagggg 4200 caccccaatc catggcctgg gttggcctca ttggctgtcc ctgaaatgct gaggaggtgg 4260 gttacttccc tccgcccagg ccagacccag gcagctgctc cccagctttc atgagcttct 4320 ttctcagatt caaacagaca gagaagaaca tggcccattc tgtgggaaga cattgcccca 4380 caggattgaa acaaaaagca acacggtgac catcaccttt gtcacagatg aatcaggaga 4440 ccacacaggc tggaagatcc actacacgag cacagtgagc aagtgggctc agatccttgg 4500 tggaagcgca gagctgcctc tctctggagt gcaaggagct gtagagtgta gggctcttct 4560 gggcaggact aggaagggac accaggttta gtggtgctga ggtctgaggc agcagcttct 4620 aaggggaagc acccgtgccc tcctcagcag cacccagcat cttcaccact cattcttcaa 4680 ccacccattc acccatcact catcttttac ccacccaccc tttgccactc atccttctgt 4740 ccctcatcct tccaaccatt catcaatcac ccacccatcc atcctttgcc acacaaccat 4800 ccacccattc ttctacctac ccatcctatc catccatcct tctatcagca tccttctacc 4860 acccatcctt cgttcggtca tccatcatca tccatccatc 4900 <210> 8 <211> 136 <212> PRT <213> Homo sapien <400> 8 Met Arg Leu Leu Thr Leu Leu Gly Leu Leu Cys Gly Ser Val Ala Thr 1 5 10 15 Pro Leu Gly Pro Lys Trp Pro Glu Pro Val Phe Gly Arg Leu Ala Ser             20 25 30 Pro Gly Phe Pro Gly Glu Tyr Ala Asn Asp Gln Glu Arg Arg Trp Thr         35 40 45 Leu Thr Ala Pro Pro Gly Tyr Arg Leu Arg Leu Tyr Phe Thr His Phe     50 55 60 Asp Leu Glu Leu Ser His Leu Cys Glu Tyr Asp Phe Val Lys Leu Ser 65 70 75 80 Ser Gly Ala Lys Val Leu Ala Thr Leu Cys Gly Gln Glu Ser Thr Asp                 85 90 95 Thr Glu Arg Ala Pro Gly Lys Asp Thr Phe Tyr Ser Leu Gly Ser Ser             100 105 110 Leu Asp Ile Thr Phe Arg Ser Asp Tyr Ser Asn Glu Lys Pro Phe Thr         115 120 125 Gly Phe Glu Ala Phe Tyr Ala Ala     130 135 <210> 9 <211> 181 <212> PRT <213> Homo sapien <400> 9 Met Arg Leu Leu Thr Leu Leu Gly Leu Leu Cys Gly Ser Val Ala Thr 1 5 10 15 Pro Leu Gly Pro Lys Trp Pro Glu Pro Val Phe Gly Arg Leu Ala Ser             20 25 30 Pro Gly Phe Pro Gly Glu Tyr Ala Asn Asp Gln Glu Arg Arg Trp Thr         35 40 45 Leu Thr Ala Pro Pro Gly Tyr Arg Leu Arg Leu Tyr Phe Thr His Phe     50 55 60 Asp Leu Glu Leu Ser His Leu Cys Glu Tyr Asp Phe Val Lys Leu Ser 65 70 75 80 Ser Gly Ala Lys Val Leu Ala Thr Leu Cys Gly Gln Glu Ser Thr Asp                 85 90 95 Thr Glu Arg Ala Pro Gly Lys Asp Thr Phe Tyr Ser Leu Gly Ser Ser             100 105 110 Leu Asp Ile Thr Phe Arg Ser Asp Tyr Ser Asn Glu Lys Pro Phe Thr         115 120 125 Gly Phe Glu Ala Phe Tyr Ala Ala Glu Asp Ile Asp Glu Cys Gln Val     130 135 140 Ala Pro Gly Glu Ala Pro Thr Cys Asp His His Cys His Asn His Leu 145 150 155 160 Gly Gly Phe Tyr Cys Ser Cys Arg Ala Gly Tyr Val Leu His Arg Asn                 165 170 175 Lys Arg Thr Cys Ser             180 <210> 10 <211> 293 <212> PRT <213> Homo sapien <400> 10 Met Arg Leu Leu Thr Leu Leu Gly Leu Leu Cys Gly Ser Val Ala Thr 1 5 10 15 Pro Leu Gly Pro Lys Trp Pro Glu Pro Val Phe Gly Arg Leu Ala Ser             20 25 30 Pro Gly Phe Pro Gly Glu Tyr Ala Asn Asp Gln Glu Arg Arg Trp Thr         35 40 45 Leu Thr Ala Pro Pro Gly Tyr Arg Leu Arg Leu Tyr Phe Thr His Phe     50 55 60 Asp Leu Glu Leu Ser His Leu Cys Glu Tyr Asp Phe Val Lys Leu Ser 65 70 75 80 Ser Gly Ala Lys Val Leu Ala Thr Leu Cys Gly Gln Glu Ser Thr Asp                 85 90 95 Thr Glu Arg Ala Pro Gly Lys Asp Thr Phe Tyr Ser Leu Gly Ser Ser             100 105 110 Leu Asp Ile Thr Phe Arg Ser Asp Tyr Ser Asn Glu Lys Pro Phe Thr         115 120 125 Gly Phe Glu Ala Phe Tyr Ala Ala Glu Asp Ile Asp Glu Cys Gln Val     130 135 140 Ala Pro Gly Glu Ala Pro Thr Cys Asp His His Cys His Asn His Leu 145 150 155 160 Gly Gly Phe Tyr Cys Ser Cys Arg Ala Gly Tyr Val Leu His Arg Asn                 165 170 175 Lys Arg Thr Cys Ser Ala Leu Cys Ser Gly Gln Val Phe Thr Gln Arg             180 185 190 Ser Gly Glu Leu Ser Ser Pro Glu Tyr Pro Arg Pro Tyr Pro Lys Leu         195 200 205 Ser Ser Cys Thr Tyr Ser Ile Ser Leu Glu Glu Gly Phe Ser Val Ile     210 215 220 Leu Asp Phe Val Glu Ser Phe Asp Val Glu Thr His Pro Glu Thr Leu 225 230 235 240 Cys Pro Tyr Asp Phe Leu Lys Ile Gln Thr Asp Arg Glu Glu His Gly                 245 250 255 Pro Phe Cys Gly Lys Thr Leu Pro His Arg Ile Glu Thr Lys Ser Asn             260 265 270 Thr Val Thr Ile Thr Phe Val Thr Asp Glu Ser Gly Asp His Thr Gly         275 280 285 Trp Lys Ile His Tyr     290 <210> 11 <211> 41 <212> PRT <213> Homo sapien <400> 11 Glu Asp Ile Asp Glu Cys Gln Val Ala Pro Gly Glu Ala Pro Thr Cys 1 5 10 15 Asp His His Cys His Asn His Leu Gly Gly Phe Tyr Cys Ser Cys Arg             20 25 30 Ala Gly Tyr Val Leu His Arg Asn Lys         35 40 <210> 12 <211> 242 <212> PRT <213> Homo sapien <400> 12 Ile Tyr Gly Gly Gln Lys Ala Lys Pro Gly Asp Phe Pro Trp Gln Val 1 5 10 15 Leu Ile Leu Gly Gly Thr Thr Ala Gly Ala Leu Leu Tyr Asp Asn             20 25 30 Trp Val Leu Thr Ala Ala His Ala Val Tyr Glu Gln Lys His Asp Ala         35 40 45 Ser Ala Leu Asp Ile Arg Met Gly Thr Leu Lys Arg Leu Ser Pro His     50 55 60 Tyr Thr Gln Ala Trp Ser Glu Ala Val Phe Ile His Glu Gly Tyr Thr 65 70 75 80 His Asp Ala Gly Phe Asp Asn Asp Ile Ala Leu Ile Lys Leu Asn Asn                 85 90 95 Lys Val Val Ile Asn Ser Asn Ile Thr Pro Ile Cys Leu Pro Arg Lys             100 105 110 Glu Ala Glu Ser Phe Met Arg Thr Asp Asp Ile Gly Thr Ala Ser Gly         115 120 125 Trp Gly Leu Thr Gln Arg Gly Phe Leu Ala Arg Asn Leu Met Tyr Val     130 135 140 Asp Ile Pro Ile Val Asp His Gln Lys Cys Thr Ala Ala Tyr Glu Lys 145 150 155 160 Pro Pro Tyr Pro Arg Gly Ser Val Thr Ala Asn Met Leu Cys Ala Gly                 165 170 175 Leu Glu Ser Gly Gly Lys Asp Ser Cys Arg Gly Asp Ser Gly Gly Ala             180 185 190 Leu Val Phe Leu Asp Ser Glu Thr Glu Arg Trp Phe Val Gly Gly Ile         195 200 205 Val Ser Trp Gly Ser Met Asn Cys Gly Glu Ala Gly Gln Tyr Gly Val     210 215 220 Tyr Thr Lys Val Ile Asn Tyr Ile Pro Trp Ile Glu Asn Ile Ile Ser 225 230 235 240 Asp Phe          <210> 13 <211> 16 <212> PRT <213> Artificial Sequence <220> <223> Synthetic peptide <400> 13 Gly Lys Asp Ser Cys Arg Gly Asp Ala Gly Gly Ala Leu Val Phe Leu 1 5 10 15 <210> 14 <211> 15 <212> PRT <213> Artificial Sequence <220> <223> Synthetic peptide <400> 14 Thr Pro Leu Gly Pro Lys Trp Pro Glu Pro Val Phe Gly Arg Leu 1 5 10 15 <210> 15 <211> 43 <212> PRT <213> Artificial Sequence <220> <223> Synthetic peptide <400> 15 Thr Ala Pro Pro Gly Tyr Arg Leu Arg Leu Tyr Phe Thr His Phe Asp 1 5 10 15 Leu Glu Leu Ser His Leu Cys Glu Tyr Asp Phe Val Lys Leu Ser Ser             20 25 30 Gly Ala Lys Val Leu Ala Thr Leu Cys Gly Gln         35 40 <210> 16 <211> 8 <212> PRT <213> Artificial <220> <223> Synthetic peptide <400> 16 Thr Phe Arg Ser Asp Tyr Ser Asn 1 5 <210> 17 <211> 25 <212> PRT <213> Artificial Sequence <220> <223> Synthetic peptide <400> 17 Phe Tyr Ser Leu Gly Ser Ser Leu Asp Ile Thr Phe Arg Ser Asp Tyr 1 5 10 15 Ser Asn Glu Lys Pro Phe Thr Gly Phe             20 25 <210> 18 <211> 9 <212> PRT <213> Artificial <220> <223> Synthetic peptide <400> 18 Ile Asp Glu Cys Gln Val Ala Pro Gly 1 5 <210> 19 <211> 25 <212> PRT <213> Artificial Sequence <220> <223> Synthetic peptide <400> 19 Ala Asn Met Leu Cys Ala Gly Leu Glu Ser Gly Gly Lys Asp Ser Cys 1 5 10 15 Arg Gly Asp Ser Gly Gly Ala Leu Val             20 25 <210> 20 <211> 960 <212> DNA <213> Homo sapien <220> <221> CDS <222> (51). (797) <400> 20 attaactgag attaaccttc cctgagtttt ctcacaccaa ggtgaggacc atg tcc 56                                                        Met Ser                                                        One ctg ttt cca tca ctc cct ctc ctt ctc ctg agt atg gtg gca gcg tct 104 Leu Phe Pro Ser Leu Pro Leu Leu Leu Leu Ser Met Val         5 10 15 tac tca gaa act gtg acc tgt gag gat gcc caa aag acc tgc cct gca 152 Tyr Ser Glu Thr Val Thr Cys Glu Asp Ala Gln Lys Thr Cys Pro Ala     20 25 30 gtg att gcc tgt agc tct cca ggc atc aac ggc ttc cca ggc aaa gat 200 Val Ile Ala Cys Ser Ser Pro Gly Ile Asn Gly Phe Pro Gly Lys Asp 35 40 45 50 ggg cgt gat ggc acc aag gga gaa aag ggg gaa cca ggc caa ggg ctc 248 Gly Arg Asp Gly Thr Lys Gly Glu Lys Gly GIy Pro Gly GInt Gly Leu                 55 60 65 aga ggc tta cag ggc ccc cct gga aag ttg ggg cct cca gga aat cca 296 Arg Gly Leu Gln Gly Pro Pro Gly Lys Leu Gly Pro Pro Gly Asn Pro             70 75 80 ggg cct tct ggg tca cca gga cca aag ggc caa aaa gga gac cct gga 344 Gly Pro Ser Gly Ser Pro Gly Pro Lys Gly Gln Lys Gly Asp Pro Gly         85 90 95 aaa agt ccg gat ggt gat agt agc ctg gct gcc tca gaa aga aaa gct 392 Lys Ser Pro Asp Gly Asp Ser Ser Leu Ala Ala Ser Glu Arg Lys Ala     100 105 110 ctg caa aca gaa atg gca cgt atc aaa aag tgg ctc acc ttc tct ctg 440 Leu Gln Thr Glu Met Ala Arg Ile Lys Lys Trp Leu Thr Phe Ser Leu 115 120 125 130 ggc aaa caa gtt ggg aac aag ttc ttc ctg acc aat ggt gaa ata atg 488 Gly Lys Gln Val Gly Asn Lys Phe Phe Leu Thr Asn Gly Glu Ile Met                 135 140 145 acc ttt gaa aaa gtg aag gcc ttg tgt gtc aag ttc cag gcc tct gtg 536 Thr Phe Glu Lys Val Lys Ala Leu Cys Val Lys Phe Gln Ala Ser Val             150 155 160 gcc acc ccc agg aat gct gca gag aat gga gcc att cag aat ctc atc 584 Ala Thr Pro Arg Asn Ala Gla Asn Gly Ala Ile Gln Asn Leu Ile         165 170 175 aag gag ga gcc ttc ctg ggc atc act gat gag aag aca gaa ggg cag 632 Lys Glu Glu Ala Phe Leu Gly Ile Thr Asp Glu Lys Thr Glu Gly Gln     180 185 190 ttt gtg gat ctg aca gga aat aga ctg acc tac aca aac tgg aac gag 680 Phe Val Asp Leu Thr Gly Asn Arg Leu Thr Tyr Thr Asn Trp Asn Glu 195 200 205 210 ggt gaa ccc aac aat gct ggt tct gat ga gat tgt gta ttg cta ctg 728 Gly Glu Pro Asn Asn Ala Gly Ser Asp Glu Asp Cys Val Leu Leu Leu                 215 220 225 aaa aat ggc cag tgg aat gac gtc ccc tgc tcc acc tcc cat ctg gcc 776 Lys Asn Gly Gln Trp Asn Asp Val Pro Cys Ser Thr Ser Leu Ala             230 235 240 gtc tgt gag ttc cct atc tga agggtcatat cactcaggcc ctccttgtct 827 Val Cys Glu Phe Pro Ile         245 ttttactgca acccacaggc ccacagtatg cttgaaaaga taaattatat caatttcctc 887 atatccagta ttgttccttt tgtgggcaat cactaaaaaat gatcactaac agcaccaaca 947 aagcaataat agt 960 <210> 21 <211> 248 <212> PRT <213> Homo sapien <400> 21 Met Ser Leu Phe Pro Ser Leu Pro Leu Leu Leu Leu Ser Met Val Ala 1 5 10 15 Ala Ser Tyr Ser Glu Thr Val Thr Cys Glu Asp Ala Gln Lys Thr Cys             20 25 30 Pro Ala Val Ile Ala Cys Ser Ser Pro Gly Ile Asn Gly Phe Pro Gly         35 40 45 Lys Asp Gly Arg Asp Gly Thr Lys Gly Glu Lys Gly Glu Pro Gly Gln     50 55 60 Gly Leu Arg Gly Leu Gln Gly Pro Pro Gly Lys Leu Gly Pro Pro Gly 65 70 75 80 Asn Pro Gly Pro Ser Gly Ser Pro Gly Pro Lys Gly Gln Lys Gly Asp                 85 90 95 Pro Gly Lys Ser Pro Asp Gly Asp Ser Ser Leu Ala Ala Ser Glu Arg             100 105 110 Lys Ala Leu Gln Thr Glu Met Ala Arg Ile Lys Lys Trp Leu Thr Phe         115 120 125 Ser Leu Gly Lys Gln Val Gly Asn Lys Phe Phe Leu Thr Asn Gly Glu     130 135 140 Ile Met Thr Phe Glu Lys Val Lys Ala Leu Cys Val Lys Phe Gln Ala 145 150 155 160 Ser Val Ala Thr Pro Arg Asn Ala Gla Asn Gly Ala Ile Gln Asn                 165 170 175 Leu Ile Lys Glu Glu Ala Phe Leu Gly Ile Thr Asp Glu Lys Thr Glu             180 185 190 Gly Gln Phe Val Asp Leu Thr Gly Asn Arg Leu Thr Tyr Thr Asn Trp         195 200 205 Asn Glu Gly Glu Pro Asn Asn Ala Gly Ser Asp Glu Asp Cys Val Leu     210 215 220 Leu Leu Lys Asn Gly Gln Trp Asn Asp Val Pro Cys Ser Thr Ser His 225 230 235 240 Leu Ala Val Cys Glu Phe Pro Ile                 245 <210> 22 <211> 6 <212> PRT <213> Artificial Sequence <220> <223> Synthetic peptide consensus sequence <220> <221> MISC_FEATURE <222> (1) <223> Wherein X at position 1 represents hydroxyproline <220> <221> MISC_FEATURE <222> (4) (4) <223> Wherein X at position 4 represents hydrophobic residue <400> 22 Xaa Gly Lys Xaa Gly Pro 1 5 <210> 23 <211> 5 <212> PRT <213> Artificial Sequence <220> <223> Synthetic peptide <220> <221> MISC_FEATURE <222> (1) <223> Wherein X represents hydroxyproline <400> 23 Xaa Gly Lys Leu Gly 1 5 <210> 24 <211> 16 <212> PRT <213> Artificial Sequence <220> <223> Synthetic peptide <220> <221> MISC_FEATURE &Lt; 222 > (9) <223> Wherein X at positions 9 and 15 represents hydroxyproline <400> 24 Gly Leu Arg Gly Leu Gln Gly Pro Xaa Gly Lys Leu Gly Pro Xaa Gly 1 5 10 15 <210> 25 <211> 27 <212> PRT <213> Artificial Sequence <220> <223> Synthetic peptide <220> <221> MISC_FEATURE <222> (3) (27) Wherein X at positions 3, 6, 15, 21, 24, 27 represent        hydroxyproline <400> 25 Gly Pro Xaa Gly Pro Xaa Gly Leu Arg Gly Leu Gln Gly Pro Xaa Gly 1 5 10 15 Lys Leu Gly Pro Xaa Gly Pro Xaa Gly Pro Xaa             20 25 <210> 26 <211> 53 <212> PRT <213> Artificial Sequence <220> <223> Synthetic peptide <220> <221> misc_feature &Lt; 222 > (26) <223> Xaa can be any naturally occurring amino acid <220> <221> misc_feature &Lt; 222 > (32) <223> Xaa can be any naturally occurring amino acid <220> <221> misc_feature &Lt; 222 > (35) <223> Xaa can be any naturally occurring amino acid <220> <221> misc_feature &Lt; 222 > (41) .. (41) <223> Xaa can be any naturally occurring amino acid <220> <221> misc_feature (50). (50) <223> Xaa can be any naturally occurring amino acid <400> 26 Gly Lys Asp Gly Arg Asp Gly Thr Lys Gly Glu Lys Gly Glu Glu Pro Gly 1 5 10 15 Gln Gly Leu Arg Gly Leu Gln Gly Pro Xaa Gly Lys Leu Gly Pro Xaa             20 25 30 Gly Asn Xaa Gly Pro Ser Gly Ser Xaa Gly Pro Lys Gly Gln Lys Gly         35 40 45 Asp Xaa Gly Lys Ser     50 <210> 27 <211> 33 <212> PRT <213> Artificial Sequence <220> <223> Synthetic peptide <220> <221> MISC_FEATURE <222> (3) (33) Wherein X at positions 3, 6, 12, 18, 21, 30, 33 represent        hydroxyproline <400> 27 Gly Ala Xaa Gly Ser Xaa Gly Glu Lys Gly Ala Xaa Gly Pro Gln Gly 1 5 10 15 Pro Xaa Gly Pro Xaa Gly Lys Met Gly Pro Lys Gly Glu Xaa Gly Asp             20 25 30 Xaa      <210> 28 <211> 45 <212> PRT <213> Artificial Sequence <220> <223> Synthetic peptide <220> <221> MISC_FEATURE <222> (3). (45) Wherein X at positions 3, 6, 9, 27, 30, 36, 42, 45 represent        hydroxyproline <400> 28 Gly Cys Xaa Gly Leu Xaa Gly Ala Xaa Gly Asp Lys Gly Glu Ala Gly 1 5 10 15 Thr Asn Gly Lys Arg Gly Glu Arg Gly Pro Xaa Gly Pro Xaa Gly Lys             20 25 30 Ala Gly Pro Xaa Gly Pro Asn Gly Ala Xaa Gly Glu Xaa         35 40 45 <210> 29 <211> 24 <212> PRT <213> Artificial Sequence <220> <223> Synthetic peptide <400> 29 Leu Gln Arg Ala Leu Glu Ile Leu Pro Asn Arg Val Thr Ile Lys Ala 1 5 10 15 Asn Arg Pro Phe Leu Val Phe Ile             20 <210> 30 <211> 559 <212> DNA <213> Homo sapien <400> 30 atgaggctgc tgaccctcct gggccttctg tgtggctcgg tggccacccc cttgggcccg 60 aagtggcctg aacctgtgtt cgggcgcctg gcatcccccg gctttccagg ggagtatgcc 120 aatgaccagg agcggcgctg gaccctgact gcaccccccg gctaccgcct gcgcctctac 180 ttcacccact tcgacctgga gctctcccac ctctgcgagt acgacttcgt caagctgagc 240 tcgggggcca aggtgctggc cacgctgtgc gggcaggaga gcacagacac ggagcgggcc 300 cctggcaagg acactttcta ctcgctgggc tccagcctgg acattacctt ccgctccgac 360 tactccaacg agaagccgtt cacggggttc gaggccttct atgcagccga ggacattgac 420 gagtgccagg tggccccggg agaggcgccc acctgcgacc accactgcca caaccacctg 480 ggcggtttct actgctcctg ccgcgcaggc tacgtcctgc accgtaacaa gcgcacctgc 540 tcagccctgt gctccggcc 559 <210> 31 <211> 34 <212> DNA <213> Artificial Sequence <220> <223> Synthetic oligo <400> 31 cgggcacacc atgaggctgc tgaccctcct gggc 34 <210> 32 <211> 33 <212> DNA <213> Artificial Sequence <220> <223> Synthetic oligo <400> 32 gacattacct tccgctccga ctccaacgag aag 33 <210> 33 <211> 33 <212> DNA <213> Artificial Sequence <220> <223> Synthetic oligo <400> 33 agcagccctg aatacccacg gccgtatccc aaa 33 <210> 34 <211> 26 <212> DNA <213> Artificial Sequence <220> <223> Synthetic oligo <400> 34 cgggatccat gaggctgctg accctc 26 <210> 35 <211> 19 <212> DNA <213> Artificial Sequence <220> <223> Synthetic oligo <400> 35 ggaattccta ggctgcata 19 <210> 36 <211> 19 <212> DNA <213> Artificial Sequence <220> <223> Synthetic oligo <400> 36 ggaattccta cagggcgct 19 <210> 37 <211> 19 <212> DNA <213> Artificial Sequence <220> <223> Synthetic oligo <400> 37 ggaattccta gtagtggat 19 <210> 38 <211> 25 <212> DNA <213> Artificial Sequence <220> <223> Synthetic oligo <400> 38 tgcggccgct gtaggtgctg tcttt 25 <210> 39 <211> 23 <212> DNA <213> Artificial Sequence <220> <223> Synthetic oligo <400> 39 ggaattcact cgttattctc gga 23 <210> 40 <211> 17 <212> DNA <213> Artificial Sequence <220> <223> Synthetic oligo <400> 40 tccgagaata acgagtg 17 <210> 41 <211> 29 <212> DNA <213> Artificial Sequence <220> <223> Synthetic oligo <400> 41 cattgaaagc tttggggtag aagttgttc 29 <210> 42 <211> 27 <212> DNA <213> Artificial Sequence <220> <223> Synthetic oligo <400> 42 cgcggccgca gctgctcaga gtgtaga 27 <210> 43 <211> 28 <212> DNA <213> Artificial Sequence <220> <223> Synthetic oligo <400> 43 cggtaagctt cactggctca gggaaata 28 <210> 44 <211> 37 <212> DNA <213> Artificial Sequence <220> <223> Synthetic oligo <400> 44 aagaagcttg ccgccaccat ggattggctg tggaact 37 <210> 45 <211> 31 <212> DNA <213> Artificial Sequence <220> <223> Synthetic oligo <400> 45 cgggatcctc aaactttctt gtccaccttg g 31 <210> 46 <211> 36 <212> DNA <213> Artificial Sequence <220> <223> Synthetic oligo <400> 46 aagaaagctt gccgccacca tgttctcact agctct 36 <210> 47 <211> 26 <212> DNA <213> Artificial Sequence <220> <223> Synthetic oligo <400> 47 cgggatcctt ctccctctaa cactct 26 <210> 48 <211> 9 <212> PRT <213> Artificial Sequence <220> <223> Synthetic peptide <400> 48 Glu Pro Lys Ser Cys Asp Lys Thr His 1 5 <210> 49 <211> 4960 <212> DNA <213> Homo Sapien <400> 49 ccggacgtgg tggcgcatgc ctgtaatccc agctactcgg gaggctgagg caggagaatt 60 gctcgaaccc cggaggcaga ggtttggtgg ctcacacctg taatcccagc actttgcgag 120 gctgaggcag gtgcatcgct ttggctcagg agttcaagac cagcctgggc aacacaggga 180 gacccccatc tctacaaaaa acaaaaacaa atataaaggg gataaaaaaa aaaaaaagac 240 aagacatgaa tccatgagga cagagtgtgg aagaggaagc agcagcctca aagttctgga 300 agctggaaga acagataaac aggtgtgaaa taactgcctg gaaagcaact tctttttttt 360 tttttttttt tttgaggtgg agtctcactc tgtcgtccag gctggagtgc agtggtgcga 420 tctcggatca ctgcaacctc cgcctcccag gctcaagcaa ttctcctgcc tcagcctccc 480 gagtagctgg gattataagt gcgcgctgcc acacctggat gatttttgta tttttagtag 540 agatgggatt tcaccatgtt ggtcaggctg gtctcaaact cccaacctcg tgatccaccc 600 accttggcct cccaaagtgc tgggattaca ggtataagcc accgagccca gccaaaagcg 660 acttctaagc ctgcaaggga atcgggaatt ggtggcacca ggtccttctg acagggttta 720 agaaattagc cagcctgagg ctgggcacgg tggctcacac ctgtaatccc agcactttgg 780 gaggctaagg caggtggatc acctgagggc aggagttcaa gaccagcctg accaacatgg 840 agaaacccca tccctaccaa aaataaaaaa ttagccaggt gtggtggtgc tcgcctgtaa 900 tcccagctac ttgggaggct gaggtgggag gattgcttga acacakgaag tagaggctgc 960 agtgagctat gattgcagca ctgcactgaa gccggggcaa cagaacaaga tccaaaaaaa 1020 agggaggggt gaggggcaga gccaggattt gtttccaggc tgttgttacc taggtccgac 1080 tcctggctcc cagagcagcc tgtcctgcct gcctggaact ctgagcaggc tggagtcatg 1140 gagtcgattc ccagaatccc agagtcaggg aggctggggg caggggcagg tcactggaca 1200 aacagatcaa aggtgagacc agcgtagggc tgcagaccag gccaggccag ctggacgggc 1260 acaccatgag gtaggtgggc gcccacagcc tccctgcagg gtgtggggtg ggagcacagg 1320 cctgggccct caccgcccct gccctgccca taggctgctg accctcctgg gccttctgtg 1380 tggctcggtg gccaccccct tgggcccgaa gtggcctgaa cctgtgttcg ggcgcctggc 1440 atcccccggc tttccagggg agtatgccaa tgaccaggag cggcgctgga ccctgactgc 1500 accccccggc taccgcctgc gcctctactt cacccacttc gacctggagc tctcccacct 1560 ctgcgagtac gacttcgtca aggtgccgtc aggacgggag ggctggggtt tctcagggtc 1620 ggggggtccc caaggagtag ccagggttca gggacacctg ggagcagggg ccaggcttgg 1680 ccaggaggga gatcaggcct gggtcttgcc ttcactccct gtgacacctg accccacagc 1740 tgagctcggg ggccaaggtg ctggccacgc tgtgcgggca ggagagcaca gacacggagc 1800 gggcccctgg caaggacact ttctactcgc tgggctccag cctggacatt accttccgct 1860 ccgactactc caacgagaag ccgttcacgg ggttcgaggc cttctatgca gccgagggtg 1920 agccaagagg ggtcctgcaa catctcagtc tgcgcagctg gctgtggggg taactctgtc 1980 ttaggccagg cagccctgcc ttcagtttcc ccacctttcc cagggcaggg gagaggcctc 2040 tggcctgaca tcatccacaa tgcaaagacc aaaacagccg tgacctccat tcacatgggc 2100 tgagtgccaa ctctgagcca gggatctgag gacagcatcg cctcaagtga cgcagggact 2160 ggccgggcgc agcagctcac gcctgtaatt ccagcacttt gggaggccga ggctggctga 2220 tcatttgagg tcaggagttc aaggccagcc agggcaacac ggtgaaactc tatctccact 2280 aaaactacaa aaattagctg ggcgtggtgg tgcgcacctg gaatcccagc tactagggag 2340 gctgaggcag gagaattgct tgaacctgcg aggtggaggc tgcagtgaac agagattgca 2400 ccactacact ccagcctggg cgacagagct agactccgtc tcaaaaaaca aaaaacaaaa 2460 acgacgcagg ggccgagggc cccatttaca gctgacaaag tggggccctg ccagcgggag 2520 cgctgccagg atgtttgatt tcagatccca gtccctgcag agaccaactg tgtgacctct 2580 ggcaagtggc tcaatttctc tgctccttag gaagctgctg caagggttca gcgctgtagc 2640 cccgccccct gggtttgatt gactcccctc attagctggg tgacctcggg ccggacactg 2700 aaactcccac tggtttaaca gaggtgatgt ttgcatcttt ctcccagcgc tgctgggagc 2760 ttgcagcgac cctaggcctg taaggtgatt ggcccggcac cagtcccgca ccctagacag 2820 gacgaggcct cctctgaggt ccactctgag gtcatggatc tcctgggagg agtccaggct 2880 ggatcccgcc tctttccctc ctgacggcct gcctggccct gcctctcccc cagacattga 2940 cgagtgccag gtggccccgg gagaggcgcc cacctgcgac caccactgcc acaaccacct 3000 gggcggtttc tactgctcct gccgcgcagg ctacgtcctg caccgtaaca agcgcacctg 3060 ctcagccctg tgctccggcc aggtcttcac ccagaggtct ggggagctca gcagccctga 3120 atacccacgg ccgtatccca aactctccag ttgcacttac agcatcagcc tggaggaggg 3180 gttcagtgtc attctggact ttgtggagtc cttcgatgtg gagacacacc ctgaaaccct 3240 gtgtccctac gactttctca agattcaaac agacagagaa gaacatggcc cattctgtgg 3300 gaagacattg ccccacagga ttgaaacaaa aagcaacacg gtgaccatca cctttgtcac 3360 agatgaatca ggagaccaca caggctggaa gatccactac acgagcacag cgcacgcttg 3420 cccttatccg atggcgccac ctaatggcca cgtttcacct gtgcaagcca aatacatcct 3480 gaaagacagc ttctccatct tttgcgagac tggctatgag cttctgcaag gtcacttgcc 3540 cctgaaatcc tttactgcag tttgtcagaa agatggatct tgggaccggc caatgcccgc 3600 gtgcagcatt gttgactgtg gccctcctga tgatctaccc agtggccgag tggagtacat 3660 cacaggtcct ggagtgacca cctacaaagc tgtgattcag tacagctgtg aagagacctt 3720 ctacacaatg aaagtgaatg atggtaaata tgtgtgtgag gctgatggat tctggacgag 3780 ctccaaagga gaaaaatcac tcccagtctg tgagcctgtt tgtggactat cagcccgcac 3840 aacaggaggg cgtatatatg gagggcaaaa ggcaaaacct ggtgattttc cttggcaagt 3900 cctgatatta ggtggaacca cagcagcagg tgcactttta tatgacaact gggtcctaac 3960 agctgctcat gccgtctatg agcaaaaaca tgatgcatcc gccctggaca ttcgaatggg 4020 caccctgaaa agactatcac ctcattatac acaagcctgg tctgaagctg tttttataca 4080 tgaaggttat actcatgatg ctggctttga caatgacata gcactgatta aattgaataa 4140 caaagttgta atcaatagca acatcacgcc tatttgtctg ccaagaaaag aagctgaatc 4200 ctttatgagg acagatgaca ttggaactgc atctggatgg ggattaaccc aaaggggttt 4260 tcttgctaga aatctaatgt atgtcgacat accgattgtt gaccatcaaa aatgtactgc 4320 tgcatatgaa aagccaccct atccaagggg aagtgtaact gctaacatgc tttgtgctgg 4380 cttagaaagt gggggcaagg acagctgcag aggtgacagc ggaggggcac tggtgtttct 4440 agatagtgaa acagagaggt ggtttgtggg aggaatagtg tcctggggtt ccatgaattg 4500 tggggaagca ggtcagtatg gagtctacac aaaagttatt aactatattc cctggatcga 4560 gaacataatt agtgattttt aacttgcgtg tctgcagtca aggattcttc atttttagaa 4620 atgcctgtga agaccttggc agcgacgtgg ctcgagaagc attcatcatt actgtggaca 4680 tggcagttgt tgctccaccc aaaaaaacag actccaggtg aggctgctgt catttctcca 4740 cttgccagtt taattccagc cttacccatt gactcaaggg gacataaacc acgagagtga 4800 cagtcatctt tgcccaccca gtgtaatgtc actgctcaaa ttacatttca ttaccttaaa 4860 aagccagtct cttttcatac tggctgttgg catttctgta aactgcctgt ccatgctctt 4920 tgtttttaaa cttgttctta ttgaaaaaaa aaaaaaaaaa 4960 <210> 50 <211> 2090 <212> DNA <213> Murine MASP-2 CDS <220> <221> CDS &Lt; 222 > (33) <400> 50 ggcgctggac tgcagagcta tggtggcaca cc atg agg cta ctc atc ttc ctg 53                                     Met Arg Leu Leu Ile Phe Leu                                     1 5 ggt ctg ctg tgg agt ttg gtg gcc aca ctt ctg ggt tca aag tgg cct 101 Gly Leu Leu Trp Ser Leu Val Ala Thr Leu Leu Gly Ser Lys Trp Pro         10 15 20 gaa cct gta ttc ggg cgc ctg gtg tcc cct ggc ttc cca gag aag tat 149 Glu Pro Val Phe Gly Arg Leu Val Ser Pro Gly Phe Pro Glu Lys Tyr     25 30 35 gct gac cat caa gat cga tcc tgg aca ctg act gca ccc cct ggc tac 197 Ala Asp His Gln Asp Arg Ser Trp Thr Leu Thr Ala Pro Pro Gly Tyr 40 45 50 55 cgc ctg cgc ctc tac ttc acc cac ttt gac ctg gaa ctc tct tac cgc 245 Arg Leu Arg Leu Tyr Phe Thr His Phe Asp Leu Glu Leu Ser Tyr Arg                 60 65 70 tgc gag tat gac ttt gtc aag ttg agc tca ggg acc aag gtg ctg gcc 293 Cys Glu Tyr Asp Phe Val Lys Leu Ser Ser Gly Thr Lys Val Leu Ala             75 80 85 aca ctg tgt ggg cag gag agt aca gac act gag cag gca cct ggc aat 341 Thr Leu Cys Gly Gln Glu Ser Thr Asp Thr Glu Gln Ala Pro Gly Asn         90 95 100 gac acc ttc tac tca ctg ggt ccc agc cta aag gtc acc ttc cac tcc 389 Asp Thr Phe Tyr Ser Leu Gly Pro Ser Leu Lys Val Thr Phe His Ser     105 110 115 gac tac tcc aat gag aag ccg ttc aca ggg ttt gag gcc ttc tat gca 437 Asp Tyr Ser Asn Glu Lys Pro Phe Thr Gly Phe Glu Ala Phe Tyr Ala 120 125 130 135 gcg gag gat gtg gat gaa tgc aga gtg tct ctg gga gac tca gtc cct 485 Ala Glu Asp Val Asp Glu Cys Arg Val Ser Leu Gly Asp Ser Val Pro                 140 145 150 tgt gac cat tat tgc cac aac tac ttg ggc ggc tac tat tgc tcc tgc 533 Cys Asp His Tyr Cys His Asn Tyr Leu Gly Gly Tyr Tyr Cys Ser Cys             155 160 165 aga gcg ggc tac att ctc cac cag aac aag cac acg tgc tca gcc ctt 581 Arg Ala Gly Tyr Ile Leu His Gln Asn Lys His Thr Cys Ser Ala Leu         170 175 180 tgt tca ggc cag gtg ttc aca gga aga tct ggg tat ctc agt agc cct 629 Cys Ser Gly Gln Val Phe Thr Gly Arg Ser Gly Tyr Leu Ser Ser Pro     185 190 195 gag tac ccg cag cca tac ccc aag ctc tcc agc tgc acc tac agc atc 677 Glu Tyr Pro Gln Pro Tyr Pro Lys Leu Ser Ser Cys Thr Tyr Ser Ile 200 205 210 215 cgc ctg gag gac ggc ttc agt gtc atc ctg gac ttc gtg gag tcc ttc 725 Arg Leu Glu Asp Gly Phe Ser Val Ile Leu Asp Phe Val Glu Ser Phe                 220 225 230 gat gtg gag acg cac cct gaa gcc cag tgc ccc tat gac tcc ctc aag 773 Asp Val Glu Thr His Pro Glu Ala Gln Cys Pro Tyr Asp Ser Leu Lys             235 240 245 ggg gaa cac ggc cca ttt tgt ggg aag acg ctg 821 Ile Gln Thr Asp Lys Gly Glu His Gly Pro Phe Cys Gly Lys Thr Leu         250 255 260 cct ccc agg att gaa act gac agc cac aag gtg acc atc acc ttt gcc 869 Pro Pro Arg Ile Glu Thr Asp Ser His Lys Val Thr Ile Thr Phe Ala     265 270 275 act gac gag tcg ggg aac cac aca ggc tgg aag ata cac tac aca agc 917 Thr Asp Glu Ser Gly Asn His Thr Gly Trp Lys Ile His Tyr Thr Ser 280 285 290 295 aca gca cgg ccc tgc cct gat cca acg gcg cca cct aat ggc agc att 965 Thr Ala Arg Pro Cys Pro Asp Pro Thr Ala Pro Pro Asn Gly Ser Ile                 300 305 310 tca cct gtg caa gcc acg taste gtc ctg aag gac agg ttt tct gtc ttc 1013 Ser Pro Val Gln Ala Thr Tyr Val Leu Lys Asp Arg Phe Ser Val Phe             315 320 325 tgc aag aca ggc ttc gag ctt ctg caa ggt tct gtc ccc ctg aaa tca 1061 Cys Lys Thr Gly Phe Glu Leu Leu Gln Gly Ser Val Pro Leu Lys Ser         330 335 340 ttc act gct gtc tgt cag aaa gat gga tct tgg gac cgg ccg atg cca 1109 Phe Thr Ala Val Cys Gln Lys Asp Gly Ser Trp Asp Arg Pro Met Pro     345 350 355 gag tgc agc att gat tgt ggc cct ccc gat gac cta ccc aat ggc 1157 Glu Cys Ser Ile Asp Cys Gly Pro Pro Asp Asp Leu Pro Asn Gly 360 365 370 375 cat gtg gac tat atc aca ggc cct caa gtg act acc tac aaa gct gtg 1205 His Val Asp Tyr Ile Thr Gly Pro Gln Val Thr Thr Tyr Lys Ala Val                 380 385 390 att cag tac agc tgt gaa gag act ttc tac aca atg agc agc aat ggt 1253 Ile Gln Tyr Ser Cys Glu Glu Thr Phe Tyr Thr Met Ser Ser Asn Gly             395 400 405 aaa tat gtg tgt gag gct gat gga ttc tgg acg agc tcc aaa gga gaa 1301 Lys Tyr Val Cys Glu Ala Asp Gly Phe Trp Thr Ser Ser Lys Gly Glu         410 415 420 aaa ctc ccc cgg gtt tgt gag cct gtt tgt ggg ctg tcc aca cac act 1349 Lys Leu Pro Pro Val Cys Glu Pro Val Cys Gly Leu Ser Thr His Thr     425 430 435 ata gga gga cgc atta gtt gga ggg cag cct gca aag cct ggt gac ttt 1397 Ile Gly Gly Arg Ile Val Gly Gly Gln Pro Ala Lys Pro Gly Asp Phe 440 445 450 455 cct tgg caa gtc ttg ttg ctg ggt caa act aca gca gca gca ggt gca 1445 Pro Trp Gln Val Leu Leu Leu Gly Gln Thr Thr Ala Ala Ala Gly Ala                 460 465 470 ctt ata cat gac aat tgg gtc cta aca gcc gct cat gct gta tat gag 1493 Leu Ile His Asp Asn Trp Val Leu Thr Ala Ala His Ala Val Tyr Glu             475 480 485 aaa aga atg gca gcg tcc tcc ctg aac atc cga atg ggc atc ctc aaa 1541 Lys Arg Met Ala Ala Ser Ser Leu Asn Ile Arg Met Gly Ile Leu Lys         490 495 500 agg ctc tca cct cat tac act caa gcc tgg ccc gag gaa atc ttt ata 1589 Arg Leu Ser Pro His Tyr Thr Gln Ala Trp Pro Glu Glu Ile Phe Ile     505 510 515 cat gaa ggc tac act cac ggt gct ggt ttt gac aat gat ata gca ttg 1637 His Glu Gly Tyr Thr His Gly Ala Gly Phe Asp Asn Asp Ile Ala Leu 520 525 530 535 att aaa ctc aag aac aaa gtc aca atac aac gga agc atc atg cct gtt 1685 Ile Lys Leu Lys Asn Lys Val Thr Ile Asn Gly Ser Ile Met Pro Val                 540 545 550 tgc cta ccg cga aaa gaa gct gca tcc tta atg aga aca gac ttc act 1733 Cys Leu Pro Arg Lys Glu Ala Ala Ser Leu Met Arg Thr Asp Phe Thr             555 560 565 gga act gtg gct ggc tgg ggg tta acc cag aag ggg ctt ctt gct aga 1781 Gly Thr Val Ala Gly Trp Gly Leu Thr Gln Lys Gly Leu Leu Ala Arg         570 575 580 aac cta atg ttt gtg gac ata cca att gct gac cac caa aaa tgt acc 1829 Asn Leu Met Phe Val Asp Ile Pro Ile Ala Asp His Gln Lys Cys Thr     585 590 595 acc gt g tat gaa aag ctc tat cca gga gta aga gta agc gct aac atg 1877 Thr Val Tyr Glu Lys Leu Tyr Pro Gly Val Arg Val Ser Ala Asn Met 600 605 610 615 ctc tgt gct ggc tta gag act ggt ggc aag gac agc tgc aga ggt gac 1925 Leu Cys Ala Gly Leu Glu Thr Gly Gly Lys Asp Ser Cys Arg Gly Asp                 620 625 630 agt ggg ggg gca tta gtg ttt cta gat aat gag aca cag cga tgg ttt 1973 Ser Gly Gly Ala Leu Val Phe Leu Asp Asn Glu Thr Gln Arg Trp Phe             635 640 645 gtg gga gga ata gtt tcc tgg ggt tcc att aat tgt ggg gcg gca ggc 2021 Val Gly Gly Ile Val Ser Trp Gly Ser Ile Asn Cys Gly Ala Ala Gly         650 655 660 cag tat ggg gtc tac aca aaa gtc atc aac tat att ccc tgg aat gag 2069 Gln Tyr Gly Val Tyr Thr Lys Val Ile Asn Tyr Ile Pro Trp Asn Glu     665 670 675 aac ata aga aat ttc taa 2090 Asn Ile Ile Ser Asn Phe 680 685 <210> 51 <211> 685 <212> PRT <213> Murine MASP-2 CDS <400> 51 Met Arg Leu Leu Ile Phe Leu Gly Leu Leu Trp Ser Leu Val Ala Thr 1 5 10 15 Leu Leu Gly Ser Lys Trp Pro Glu Pro Val Phe Gly Arg Leu Val Ser             20 25 30 Pro Gly Phe Pro Glu Lys Tyr Ala Asp His Gln Asp Arg Ser Trp Thr         35 40 45 Leu Thr Ala Pro Pro Gly Tyr Arg Leu Arg Leu Tyr Phe Thr His Phe     50 55 60 Asp Leu Glu Leu Ser Tyr Arg Cys Glu Tyr Asp Phe Val Lys Leu Ser 65 70 75 80 Ser Gly Thr Lys Val Leu Ala Thr Leu Cys Gly Gln Glu Ser Thr Asp                 85 90 95 Thr Glu Gln Ala Pro Gly Asn Asp Thr Phe Tyr Ser Leu Gly Pro Ser             100 105 110 Leu Lys Val Thr Phe His Ser Asp Tyr Ser Asn Glu Lys Pro Phe Thr         115 120 125 Gly Phe Glu Ala Phe Tyr Ala Ala Glu Asp Val Asp Glu Cys Arg Val     130 135 140 Ser Leu Gly Asp Ser Val Pro Cys Asp His Tyr Cys His Asn Tyr Leu 145 150 155 160 Gly Gly Tyr Tyr Cys Ser Cys Arg Ala Gly Tyr Ile Leu His Gln Asn                 165 170 175 Lys His Thr Cys Ser Ala Leu Cys Ser Gly Gln Val Phe Thr Gly Arg             180 185 190 Ser Gly Tyr Leu Ser Ser Pro Glu Tyr Pro Gln Pro Tyr Pro Lys Leu         195 200 205 Ser Ser Cys Thr Tyr Ser Ile Arg Leu Glu Asp Gly Phe Ser Val Ile     210 215 220 Leu Asp Phe Val Glu Ser Phe Asp Val Glu Thr His Pro Glu Ala Gln 225 230 235 240 Cys Pro Tyr Asp Ser Leu Lys Ile Gln Thr Asp Lys Gly Glu His Gly                 245 250 255 Pro Phe Cys Gly Lys Thr Leu Pro Pro Arg Ile Glu Thr Asp Ser His             260 265 270 Lys Val Thr Ile Thr Phe Ala Thr Asp Glu Ser Gly Asn His Thr Gly         275 280 285 Trp Lys Ile His Tyr Thr Ser Thr Ala Arg Pro Cys Pro Asp Pro Thr     290 295 300 Ala Pro Pro Asn Gly Ser Ile Ser Pro Val Gln Ala Thr Tyr Val Leu 305 310 315 320 Lys Asp Arg Phe Ser Val Phe Cys Lys Thr Gly Phe Glu Leu Leu Gln                 325 330 335 Gly Ser Val Pro Leu Lys Ser Phe Thr Ala Val Cys Gln Lys Asp Gly             340 345 350 Ser Trp Asp Arg Pro Met Pro Glu Cys Ser Ile Asp Cys Gly Pro         355 360 365 Pro Asp Asp Leu Pro Asn Gly His Val Asp Tyr Ile Thr Gly Pro Gln     370 375 380 Val Thr Thr Tyr Lys Ala Val Ile Gln Tyr Ser Cys Glu Glu Thr Phe 385 390 395 400 Tyr Thr Met Ser Ser Asn Gly Lys Tyr Val Cys Glu Ala Asp Gly Phe                 405 410 415 Trp Thr Ser Ser Lys Gly Glu Lys Leu Pro Pro Val Cys Glu Pro Val             420 425 430 Cys Gly Leu Ser Thr His Thr Ile Gly Gly Arg Ile Val Gly Gly Gln         435 440 445 Pro Ala Lys Pro Gly Asp Phe Pro Trp Gln Val Leu Leu Leu Gly Gln     450 455 460 Thr Thr Ala Ala Gly Ala Leu Ile His Asp Asn Trp Val Leu Thr 465 470 475 480 Ala Ala His Ala Val Tyr Ala Ala Ser Ser Leu Asn                 485 490 495 Ile Arg Met Gly Ile Leu Lys Arg Leu Ser Pro His Tyr Thr Gln Ala             500 505 510 Trp Pro Glu Glu Ile Phe Ile His Glu Gly Tyr Thr His Gly Ala Gly         515 520 525 Phe Asp Asn Asp Ile Ala Leu Ile Lys Leu Lys Asn Lys Val Thr Ile     530 535 540 Asn Gly Ser Ile Met Pro Val Cys Leu Pro Arg Lys Glu Ala Ala Ser 545 550 555 560 Leu Met Arg Thr Asp Phe Thr Gly Thr Val Ala Gly Trp Gly Leu Thr                 565 570 575 Gln Lys Gly Leu Leu Ala Arg Asn Leu Met Phe Val Asp Ile Pro Ile             580 585 590 Ala Asp His Gln Lys Cys Thr Thr Val Tyr Glu Lys Leu Tyr Pro Gly         595 600 605 Val Arg Val Ser Ala Asn Met Leu Cys Ala Gly Leu Glu Thr Gly Gly     610 615 620 Lys Asp Ser Cys Arg Gly Asp Ser Gly Gly Ala Leu Val Phe Leu Asp 625 630 635 640 Asn Glu Thr Gln Arg Trp Phe Val Gly Gly Ile Val Ser Trp Gly Ser                 645 650 655 Ile Asn Cys Gly Ala Ala Gly Gln Tyr Gly Val Tyr Thr Lys Val Ile             660 665 670 Asn Tyr Ile Pro Trp Asn Glu Asn Ile Ile Ser Asn Phe         675 680 685 <210> 52 <211> 670 <212> PRT <213> Murine MASP-2 mature protein <400> 52 Thr Leu Leu Gly Ser Lys Trp Pro Glu Pro Val Phe Gly Arg Leu Val 1 5 10 15 Ser Pro Gly Phe Pro Glu Lys Tyr Ala Asp His Gln Asp Arg Ser Trp             20 25 30 Thr Leu Thr Ala Pro Pro Gly Tyr Arg Leu Arg Leu Tyr Phe Thr His         35 40 45 Phe Asp Leu Glu Leu Ser Tyr Arg Cys Glu Tyr Asp Phe Val Lys Leu     50 55 60 Ser Ser Gly Thr Lys Val Leu Ala Thr Leu Cys Gly Gln Glu Ser Thr 65 70 75 80 Asp Thr Glu Gln Ala Pro Gly Asn Asp Thr Phe Tyr Ser Leu Gly Pro                 85 90 95 Ser Leu Lys Val Thr Phe His Ser Asp Tyr Ser Asn Glu Lys Pro Phe             100 105 110 Thr Gly Phe Glu Ala Phe Tyr Ala Ala Glu Asp Val Asp Glu Cys Arg         115 120 125 Val Ser Leu Gly Asp Ser Val Pro Cys Asp His Tyr Cys His Asn Tyr     130 135 140 Leu Gly Gly Tyr Tyr Cys Ser Cys Arg Ala Gly Tyr Ile Leu His Gln 145 150 155 160 Asn Lys His Thr Cys Ser Ala Leu Cys Ser Gly Gln Val Phe Thr Gly                 165 170 175 Arg Ser Gly Tyr Leu Ser Ser Pro Glu Tyr Pro Gln Pro Tyr Pro Lys             180 185 190 Leu Ser Ser Cys Thr Tyr Ser Ile Arg Leu Glu Asp Gly Phe Ser Val         195 200 205 Ile Leu Asp Phe Val Glu Ser Phe Asp Val Glu Thr His Pro Glu Ala     210 215 220 Gln Cys Pro Tyr Asp Ser Leu Lys Ile Gln Thr Asp Lys Gly Glu His 225 230 235 240 Gly Pro Phe Cys Gly Lys Thr Leu Pro Pro Arg Ile Glu Thr Asp Ser                 245 250 255 His Lys Val Thr Ile Thr Phe Ala Thr Asp Glu Ser Gly Asn His Thr             260 265 270 Gly Trp Lys Ile His Tyr Thr Ser Thr Ala Arg Pro Cys Pro Asp Pro         275 280 285 Thr Ala Pro Pro Asn Gly Ser Ile Ser Pro Val Gln Ala Thr Tyr Val     290 295 300 Leu Lys Asp Arg Phe Ser Val Phe Cys Lys Thr Gly Phe Glu Leu Leu 305 310 315 320 Gln Gly Ser Val Pro Leu Lys Ser Phe Thr Ala Val Cys Gln Lys Asp                 325 330 335 Gly Ser Trp Asp Arg Pro Met Pro Glu Cys Ser Ile Asp Cys Gly             340 345 350 Pro Pro Asp Leu Pro Asn Gly His Val Asp Tyr Ile Thr Gly Pro         355 360 365 Gln Val Thr Thr Tyr Lys Ala Val Ile Gln Tyr Ser Cys Glu Glu Thr     370 375 380 Phe Tyr Thr Met Ser Ser Asn Gly Lys Tyr Val Cys Glu Ala Asp Gly 385 390 395 400 Phe Trp Thr Ser Ser Lys Gly Glu Lys Leu Pro Pro Val Cys Glu Pro                 405 410 415 Val Cys Gly Leu Ser Thr His Thr Ile Gly Gly Arg Ile Val Gly Gly             420 425 430 Gln Pro Ala Lys Pro Gly Asp Phe Pro Trp Gln Val Leu Leu Leu Gly         435 440 445 Gln Thr Thr Ala Ala Gly Ala Leu Ile His Asp Asn Trp Val Leu     450 455 460 Thr Ala Ala His Ala Val Tyr Glu Lys Arg Ala Ala Ser Ser Leu 465 470 475 480 Asn Ile Arg Met Gly Ile Leu Lys Arg Leu Ser Pro His Tyr Thr Gln                 485 490 495 Ala Trp Pro Glu Glu Ile Phe Ile His Glu Gly Tyr Thr His Gly Ala             500 505 510 Gly Phe Asp Asn Asp Ile Ala Leu Ile Lys Leu Lys Asn Lys Val Thr         515 520 525 Ile Asn Gly Ser Ile Met Pro Val Cys Leu Pro Arg Lys Glu Ala Ala     530 535 540 Ser Leu Met Arg Thr Asp Phe Thr Gly Thr Val Ala Gly Trp Gly Leu 545 550 555 560 Thr Gln Lys Gly Leu Ala Arg Asn Leu Met Phe Val Asp Ile Pro                 565 570 575 Ile Ala Asp His Gln Lys Cys Thr Thr Val Tyr Glu Lys Leu Tyr Pro             580 585 590 Gly Val Val Ser Ala Asn Met Leu Cys Ala Gly Leu Glu Thr Gly         595 600 605 Gly Lys Asp Ser Cys Arg Gly Asp Ser Gly Gly Ala Leu Val Phe Leu     610 615 620 Asp Asn Glu Thr Gln Arg Trp Phe Val Gly Gly Ile Val Ser Trp Gly 625 630 635 640 Ser Ile Asn Cys Gly Ala Ala Gly Gln Tyr Gly Val Tyr Thr Lys Val                 645 650 655 Ile Asn Tyr Ile Pro Trp Asn Glu Asn Ile Ile Ser Asn Phe             660 665 670 <210> 53 <211> 2091 <212> DNA <213> Rat MASP-2 CDS <220> <221> CDS &Lt; 222 > (10). (2067) <400> 53 tggcacaca atg agg ct ctg atc gtc ctg ggt ctg ctt tgg agt ttg gtg 51           Met Arg Leu Leu Ile Val Leu Gly Leu Leu Trp Ser Leu Val           1 5 10 gcc aca ctt ttg ggc tcc aag tgg cct gag cct gta ttc ggg cgc ctg 99 Ala Thr Leu Leu Gly Ser Lys Trp Pro Glu Pro Val Phe Gly Arg Leu 15 20 25 30 gtg tcc ctg gcc ttc cca gag aag tat ggc aac cat cag gat cga tcc 147 Val Ser Leu Ala Phe Pro Glu Lys Tyr Gly Asn His Gln Asp Arg Ser                 35 40 45 tgg acg ctg act gca ccc cct ggc ttc cgc ctg cgc ctc tac ttc acc 195 Trp Thr Leu Thr Ala Pro Pro Gly Phe Arg Leu Arg Leu Tyr Phe Thr             50 55 60 cac ttc aac ctg gaa ctc tct tac cgc tgc gag tat gac ttt gtc aag 243 His Phe Asn Leu Glu Leu Ser Tyr Arg Cys Glu Tyr Asp Phe Val Lys         65 70 75 ttg acc tca ggg acc aag gtg cta gcc acg ctg tgt ggg cag gag agt 291 Leu Thr Ser Gly Thr Lys Val Leu Ala Thr Leu Cys Gly Gln Glu Ser     80 85 90 aca gat act gag cgg gca cct ggc aat gac acc ttc tac tca ctg ggt 339 Thr Asp Thr Glu Arg Ala Pro Gly Asn Asp Thr Phe Tyr Ser Leu Gly 95 100 105 110 ccc agc cta aag gtc acc ttc cac tcc gac tac tcc aat gag aag cca 387 Pro Ser Leu Lys Val Thr Phe His Ser Asp Tyr Ser Asn Glu Lys Pro                 115 120 125 ttc aca gga ttt gag gcc ttc tat gca gcg gag gat gtg gat gaa tgc 435 Phe Thr Gly Phe Glu Ala Phe Tyr Ala Ala Glu Asp Val Asp Glu Cys             130 135 140 aga aca tcc ctg gga gac tca gtc cct tgt gac cat tat tgc cac aac 483 Arg Thr Ser Leu Gly Asp Ser Val Pro Cys Asp His Tyr Cys His Asn         145 150 155 tac ctg ggc ggc tac tac tgc tcc tgc cga gtg ggc tac att ctg cac 531 Tyr Leu Gly Gly Tyr Tyr Cys Ser Cys Arg Val Gly Tyr Ile Leu His     160 165 170 cag aac aag cat acc tgc tca gcc ctt tgt tca ggc cag gtg ttc act 579 Gln Asn Lys His Thr Cys Ser Ala Leu Cys Ser Gly Gln Val Phe Thr 175 180 185 190 ggg agg tct ggc ttt ctc agt agc cct gag tac cca cag cca tac ccc 627 Gly Arg Ser Gly Phe Leu Ser Ser Pro Glu Tyr Pro Gln Pro Tyr Pro                 195 200 205 aaa ctc tcc agc tgc gcc tac aac atc cgc ctg gag gaa ggc ttc agt 675 Lys Leu Ser Ser Cys Ala Tyr Asn Ile Arg Leu Glu Glu Gly Phe Ser             210 215 220 atc acc ctg gac ttc gtg gag tcc ttt gat gtg gag atg cac cct gaa 723 Ile Thr Leu Asp Phe Val Glu Ser Phe Asp Val Glu Met His Pro Glu         225 230 235 gcc cag tgc ccc tac gac tcc ctc aag att caa aca gac aag agg gaa 771 Ala Gln Cys Pro Tyr Asp Ser Leu Lys Ile Gln Thr Asp Lys Arg Glu     240 245 250 tac ggc ccg ttt tgt ggg aag acg ctg ccc ccc agg att gaa act gac 819 Tyr Gly Pro Phe Cys Gly Lys Thr Leu Pro Pro Arg Ile Glu Thr Asp 255 260 265 270 agc aac aag gtg acc acc acc ttt acc acc gac gag tca ggg aac cac 867 Ser Asn Lys Val Thr Ile Thr Phe Thr Thr Asp Glu Ser Gly Asn His                 275 280 285 aca ggc tgg aag ata cac tac aca agc aca gca cag ccc tgc cct gat 915 Thr Gly Trp Lys Ile His Tyr Thr Ser Thr Ala Gln Pro Cys Pro Asp             290 295 300 cca acg gcg cca cct aat ggt cac att tca cct gtg caa gcc acg tat 963 Pro Thr Ala Pro Pro Asn Gly His Ile Ser Pro Val Gln Ala Thr Tyr         305 310 315 gtc ctg aag gac agc ttt tct gtc ttc tgc aag act ggc ttc gag ctt 1011 Val Leu Lys Asp Ser Phe Ser Val Phe Cys Lys Thr Gly Phe Glu Leu     320 325 330 ctg caa ggt tct gtc ccc ctg aag tca ttc act gct gtc tgt cag aaa 1059 Leu Gln Gly Ser Val Pro Leu Lys Ser Phe Thr Ala Val Cys Gln Lys 335 340 345 350 gat gga tct tgg gac cgg ccg ata cca gag tgc agc att att gac tgt 1107 Asp Gly Ser Trp Asp Arg Pro Ile Pro Glu Cys Ser Ile Asp Cys                 355 360 365 ggc cct ccc gat cac ccc ccc aat ggc cac gtg gac tat atc aca ggc 1155 Gly Pro Pro Asp Asp Leu Pro Asn Gly His Val Asp Tyr Ile Thr Gly             370 375 380 cct gaa gtg acc acc tac aaa gct gtg att cag tac agc tgt gaa gag 1203 Pro Glu Val Thr Thr Tyr Lys Ala Val Ile Gln Tyr Ser Cys Glu Glu         385 390 395 act ttc tac aca atg agc agc aat ggt aaa tat gtg tgt gag gct gat 1251 Thr Phe Tyr Thr Met Ser Ser Asn Gly Lys Tyr Val Cys Glu Ala Asp     400 405 410 gga ttc tgg acg agc tcc aaa gga gaa aaa tcc ctc ccg gtt tgc aag 1299 Gly Phe Trp Thr Ser Ser Lys Gly Glu Lys Ser Leu Pro Val Cys Lys 415 420 425 430 cct gtc tgt gga ctg tcc aca cac act tca gga ggc cgt ata att gga 1347 Pro Val Cys Gly Leu Ser Thr His Thr Ser Gly Gly Arg Ile Ile Gly                 435 440 445 gga cag cct gca aag cct ggt gac ttt cct tgg caa gtc ttg tta ctg 1395 Gly Gln Pro Ala Lys Pro Gly Asp Phe Pro Trp Gln Val Leu Leu Leu             450 455 460 ggt gaa act aca gca gca ggt gct ctt ata cat gac gac tgg gtc cta 1443 Gly Glu Thr Thr Ala Ala Gly Ala Leu Ile His Asp Asp Trp Val Leu         465 470 475 aca gcg gct cat gct gta tat ggg aaa aca gag gcg atg tcc tcc ctg 1491 Thr Ala His Ala Val Tyr Gly Lys Thr Glu Ala Met Ser Ser Leu     480 485 490 gac atc cgc atg ggc atc ctc aaa agg ctc tcc ctc att tac act caa 1539 Asp Ile Arg Met Gly Ile Leu Lys Arg Leu Ser Leu Ile Tyr Thr Gln 495 500 505 510 gcc tgg cca gag gct gtc ttt atc cat gaa ggc tac act cac gga gct 1587 Ala Trp Pro Glu Ala Val Phe Ile His Glu Gly Tyr Thr His Gly Ala                 515 520 525 ggt ttt gac aat gat ata gca ctg att aaa ctc aag aac aaa gtc aca 1635 Gly Phe Asp Asn Asp Ile Ala Leu Ile Lys Leu Lys Asn Lys Val Thr             530 535 540 atc aac aga aac atc atg ccg att tgt cta cca aga aaa gaa gct gca 1683 Ile Asn Arg Asn Ile Met Pro Ile Cys Leu Pro Arg Lys Glu Ala Ala         545 550 555 tcc tta atg aaa aca gac ttc gtt gga act gtg gct ggc tgg ggg tta 1731 Ser Leu Met Lys Thr Asp Phe Val Gly Thr Val Ala Gly Trp Gly Leu     560 565 570 acc cag aag ggg ttt ctt gct aga aac cta atg ttt gtg gac ata cca 1779 Thr Gln Lys Gly Phe Leu Ala Arg Asn Leu Met Phe Val Asp Ile Pro 575 580 585 590 att gtt gac cac caa aaa tgt gct act gcg tat aca aag cag ccc tac 1827 Ile Val Asp His Gln Lys Cys Ala Thr Ala Tyr Thr Lys Gln Pro Tyr                 595 600 605 cca gga gca aaa gtg act gtt aac atg ctc tgt gct ggc cta gac cgc 1875 Pro Gly Ala Lys Val Thr Val Asn Met Leu Cys Ala Gly Leu Asp Arg             610 615 620 ggt ggc aag gac agc tgc aga ggt gac agc gga ggg gca tta gtg ttt 1923 Gly Gly Lys Asp Ser Cys Arg Gly Asp Ser Gly Gly Ala Leu Val Phe         625 630 635 cta gac aat gaa aca cag aga tgg ttt gtg gga gga ata gtt tcc tgg 1971 Leu Asp Asn Glu Thr Gln Arg Trp Phe Val Gly Gly Ile Val Ser Trp     640 645 650 ggt tct aac tgt ggg ggg tca gaa cag tat ggg gtc tac acg aaa 2019 Gly Ser Ile Asn Cys Gly Gly Ser Glu Gln Tyr Gly Val Tyr Thr Lys 655 660 665 670 gtc acg aac tat ccc tgg att gag aac ata aa aat ttc taa 2067 Val Thr Asn Tyr Ile Pro Trp Ile Glu Asn Ile Ile Asn Asn Phe                 675 680 685 tttgcaaaaa aaaaaaaaaa aaaa 2091 <210> 54 <211> 685 <212> PRT <213> Rat MASP-2 CDS <400> 54 Met Arg Leu Leu Ile Val Leu Gly Leu Leu Trp Ser Leu Val Ala Thr 1 5 10 15 Leu Leu Gly Ser Lys Trp Pro Glu Pro Val Phe Gly Arg Leu Val Ser             20 25 30 Leu Ala Phe Pro Glu Lys Tyr Gly Asn His Gln Asp Arg Ser Trp Thr         35 40 45 Leu Thr Ala Pro Pro Gly Phe Arg Leu Arg Leu Tyr Phe Thr His Phe     50 55 60 Asn Leu Glu Leu Ser Tyr Arg Cys Glu Tyr Asp Phe Val Lys Leu Thr 65 70 75 80 Ser Gly Thr Lys Val Leu Ala Thr Leu Cys Gly Gln Glu Ser Thr Asp                 85 90 95 Thr Glu Arg Ala Pro Gly Asn Asp Thr Phe Tyr Ser Leu Gly Pro Ser             100 105 110 Leu Lys Val Thr Phe His Ser Asp Tyr Ser Asn Glu Lys Pro Phe Thr         115 120 125 Gly Phe Glu Ala Phe Tyr Ala Ala Glu Asp Val Asp Glu Cys Arg Thr     130 135 140 Ser Leu Gly Asp Ser Val Pro Cys Asp His Tyr Cys His Asn Tyr Leu 145 150 155 160 Gly Gly Tyr Tyr Cys Ser Cys Arg Val Gly Tyr Ile Leu His Gln Asn                 165 170 175 Lys His Thr Cys Ser Ala Leu Cys Ser Gly Gln Val Phe Thr Gly Arg             180 185 190 Ser Gly Phe Leu Ser Ser Pro Glu Tyr Pro Gln Pro Tyr Pro Lys Leu         195 200 205 Ser Ser Cys Ala Tyr Asn Ile Arg Leu Glu Glu Gly Phe Ser Ile Thr     210 215 220 Leu Asp Phe Val Glu Ser Phe Asp Val Glu Met His Pro Glu Ala Gln 225 230 235 240 Cys Pro Tyr Asp Ser Leu Lys Ile Gln Thr Asp Lys Arg Glu Tyr Gly                 245 250 255 Pro Phe Cys Gly Lys Thr Leu Pro Pro Arg Ile Glu Thr Asp Ser Asn             260 265 270 Lys Val Thr Ile Thr Phe Thr Thr Asp Glu Ser Gly Asn His Thr Gly         275 280 285 Trp Lys Ile His Tyr Thr Ser Thr Ala Gln Pro Cys Pro Asp Pro Thr     290 295 300 Ala Pro Pro Asn Gly His Ile Ser Pro Val Gln Ala Thr Tyr Val Leu 305 310 315 320 Lys Asp Ser Phe Ser Val Phe Cys Lys Thr Gly Phe Glu Leu Leu Gln                 325 330 335 Gly Ser Val Pro Leu Lys Ser Phe Thr Ala Val Cys Gln Lys Asp Gly             340 345 350 Ser Trp Asp Arg Pro Ile Pro Glu Cys Ser Ile Asp Cys Gly Pro         355 360 365 Pro Asp Asp Leu Pro Asn Gly His Val Asp Tyr Ile Thr Gly Pro Glu     370 375 380 Val Thr Thr Tyr Lys Ala Val Ile Gln Tyr Ser Cys Glu Glu Thr Phe 385 390 395 400 Tyr Thr Met Ser Ser Asn Gly Lys Tyr Val Cys Glu Ala Asp Gly Phe                 405 410 415 Trp Thr Ser Ser Lys Gly Glu Lys Ser Leu Pro Val Cys Lys Pro Val             420 425 430 Cys Gly Leu Ser Thr His Thr Ser Gly Gly Arg Ile Ile Gly Gly Gln         435 440 445 Pro Ala Lys Pro Gly Asp Phe Pro Trp Gln Val Leu Leu Leu Gly Glu     450 455 460 Thr Thr Ala Gla Ala Leu Ile His Asp Asp Trp Val Leu Thr Ala 465 470 475 480 Ala His Ala Val Tyr Gly Lys Thr Glu Ala Met Ser Ser Leu Asp Ile                 485 490 495 Arg Met Gly Ile Leu Lys Arg Leu Ser Leu Ile Tyr Thr Gln Ala Trp             500 505 510 Pro Glu Ala Val Phe Ile His Glu Gly Tyr Thr His Gly Ala Gly Phe         515 520 525 Asp Asn Asp Ile Ala Leu Ile Lys Leu Lys Asn Lys Val Thr Ile Asn     530 535 540 Arg Asn Ile Met Pro Ile Cys Leu Pro Arg Lys Glu Ala Ala Ser Leu 545 550 555 560 Met Lys Thr Asp Phe Val Gly Thr Val Ala Gly Trp Gly Leu Thr Gln                 565 570 575 Lys Gly Phe Leu Ala Arg Asn Leu Met Phe Val Asp Ile Pro Ile Val             580 585 590 Asp His Gln Lys Cys Ala Thr Ala Tyr Thr Lys Gln Pro Tyr Pro Gly         595 600 605 Ala Lys Val Thr Val Asn Met Leu Cys Ala Gly Leu Asp Arg Gly Gly     610 615 620 Lys Asp Ser Cys Arg Gly Asp Ser Gly Gly Ala Leu Val Phe Leu Asp 625 630 635 640 Asn Glu Thr Gln Arg Trp Phe Val Gly Gly Ile Val Ser Trp Gly Ser                 645 650 655 Ile Asn Cys Gly Gly Ser Glu Gln Tyr Gly Val Tyr Thr Lys Val Thr             660 665 670 Asn Tyr Ile Pro Trp Ile Glu Asn Ile Ile Asn Asn Phe         675 680 685 <210> 55 <211> 670 <212> PRT <213> Rat MASP-2 mature protein <400> 55 Thr Leu Leu Gly Ser Lys Trp Pro Glu Pro Val Phe Gly Arg Leu Val 1 5 10 15 Ser Leu Ala Phe Pro Glu Lys Tyr Gly Asn His Gln Asp Arg Ser Trp             20 25 30 Thr Leu Thr Ala Pro Pro Gly Phe Arg Leu Arg Leu Tyr Phe Thr His         35 40 45 Phe Asn Leu Glu Leu Ser Tyr Arg Cys Glu Tyr Asp Phe Val Lys Leu     50 55 60 Thr Ser Gly Thr Lys Val Leu Ala Thr Leu Cys Gly Gln Glu Ser Thr 65 70 75 80 Asp Thr Glu Arg Ala Pro Gly Asn Asp Thr Phe Tyr Ser Leu Gly Pro                 85 90 95 Ser Leu Lys Val Thr Phe His Ser Asp Tyr Ser Asn Glu Lys Pro Phe             100 105 110 Thr Gly Phe Glu Ala Phe Tyr Ala Ala Glu Asp Val Asp Glu Cys Arg         115 120 125 Thr Ser Leu Gly Asp Ser Val Pro Cys Asp His Tyr Cys His Asn Tyr     130 135 140 Leu Gly Gly Tyr Tyr Cys Ser Cys Arg Val Gly Tyr Ile Leu His Gln 145 150 155 160 Asn Lys His Thr Cys Ser Ala Leu Cys Ser Gly Gln Val Phe Thr Gly                 165 170 175 Arg Ser Gly Phe Leu Ser Ser Pro Glu Tyr Pro Gln Pro Tyr Pro Lys             180 185 190 Leu Ser Ser Cys Ala Tyr Asn Ile Arg Leu Glu Glu Gly Phe Ser Ile         195 200 205 Thr Leu Asp Phe Val Glu Ser Phe Asp Val Glu Met His Pro Glu Ala     210 215 220 Gln Cys Pro Tyr Asp Ser Leu Lys Ile Gln Thr Asp Lys Arg Glu Tyr 225 230 235 240 Gly Pro Phe Cys Gly Lys Thr Leu Pro Pro Arg Ile Glu Thr Asp Ser                 245 250 255 Asn Lys Val Thr Ile Thr Phe Thr Thr Asp Glu Ser Gly Asn His Thr             260 265 270 Gly Trp Lys Ile His Tyr Thr Ser Thr Ala Gln Pro Cys Pro Asp Pro         275 280 285 Thr Ala Pro Pro Asn Gly His Ile Ser Pro Val Gln Ala Thr Tyr Val     290 295 300 Leu Lys Asp Ser Phe Ser Val Phe Cys Lys Thr Gly Phe Glu Leu Leu 305 310 315 320 Gln Gly Ser Val Pro Leu Lys Ser Phe Thr Ala Val Cys Gln Lys Asp                 325 330 335 Gly Ser Trp Asp Arg Pro Ile Pro Glu Cys Ser Ile Asp Cys Gly             340 345 350 Pro Pro Asp Leu Pro Asn Gly His Val Asp Tyr Ile Thr Gly Pro         355 360 365 Glu Val Thr Thr Tyr Lys Ala Val Ile Gln Tyr Ser Cys Glu Glu Thr     370 375 380 Phe Tyr Thr Met Ser Ser Asn Gly Lys Tyr Val Cys Glu Ala Asp Gly 385 390 395 400 Phe Trp Thr Ser Ser Lys Gly Glu Lys Ser Leu Pro Val Cys Lys Pro                 405 410 415 Val Cys Gly Leu Ser Thr His Thr Ser Gly Gly Arg Ile Ile Gly Gly             420 425 430 Gln Pro Ala Lys Pro Gly Asp Phe Pro Trp Gln Val Leu Leu Leu Gly         435 440 445 Glu Thr Thr Ala Gla Ala Leu Ile His Asp Asp Trp Val Leu Thr     450 455 460 Ala Ala His Ala Val Tyr Gly Lys Thr Glu Ala Met Ser Ser Leu Asp 465 470 475 480 Ile Arg Met Gly Ile Leu Lys Arg Leu Ser Leu Ile Tyr Thr Gln Ala                 485 490 495 Trp Pro Glu Ala Val Phe Ile His Glu Gly Tyr Thr His Gly Ala Gly             500 505 510 Phe Asp Asn Asp Ile Ala Leu Ile Lys Leu Lys Asn Lys Val Thr Ile         515 520 525 Asn Arg Asn Ile Met Pro Ile Cys Leu Pro Arg Lys Glu Ala Ala Ser     530 535 540 Leu Met Lys Thr Asp Phe Val Gly Thr Val Ala Gly Trp Gly Leu Thr 545 550 555 560 Gln Lys Gly Phe Leu Ala Arg Asn Leu Met Phe Val Asp Ile Pro Ile                 565 570 575 Val Asp His Gln Lys Cys Ala Thr Ala Tyr Thr Lys Gln Pro Tyr Pro             580 585 590 Gly Ala Lys Val Thr Val Asn Met Leu Cys Ala Gly Leu Asp Arg Gly         595 600 605 Gly Lys Asp Ser Cys Arg Gly Asp Ser Gly Gly Ala Leu Val Phe Leu     610 615 620 Asp Asn Glu Thr Gln Arg Trp Phe Val Gly Gly Ile Val Ser Trp Gly 625 630 635 640 Ser Ile Asn Cys Gly Gly Ser Glu Gln Tyr Gly Val Tyr Thr Lys Val                 645 650 655 Thr Asn Tyr Ile Pro Trp Ile Glu Asn Ile Ile Asn Asn Phe             660 665 670 <210> 56 <211> 28 <212> DNA <213> Artificial Sequence <220> <223> Homo Sapien MASP-2 PCR primer <400> 56 atgaggctgc tgaccctcct gggccttc 28 <210> 57 <211> 23 <212> DNA <213> Artificial Sequence <220> <223> Homo Sapien MASP-2 PCR primer <400> 57 gtgcccctcc tgcgtcacct ctg 23 <210> 58 <211> 23 <212> DNA <213> Artificial Sequence <220> <223> Homo Sapien MASP-2 PCR primer <400> 58 cagaggtgac gcaggagggg cac 23 <210> 59 <211> 27 <212> DNA <213> Artificial Sequence <220> <223> Homo Sapien MASP-2 PCR primer <400> 59 ttaaaatcac taattatgtt ctcgatc 27 <210> 60 <211> 22 <212> DNA <213> Artificial Sequence <220> <223> Murine MASP-2 PCR primer <400> 60 atgaggctac tcatcttcct gg 22 <210> 61 <211> 23 <212> DNA <213> Artificial Sequence <220> <223> Murine MASP-2 PCR primer <400> 61 ctgcagaggt gacgcagggg ggg 23 <210> 62 <211> 23 <212> DNA <213> Artificial Sequence <220> <223> Murine MASP-2 PCR primer <400> 62 ccccccctgc gtcacctctg cag 23 <210> 63 <211> 29 <212> DNA <213> Artificial Sequence <220> <223> Murine MASP-2 PCR primer <400> 63 ttagaaatta cttattatgt tctcaatcc 29 <210> 64 <211> 29 <212> DNA <213> Artificial Sequence <220> <223> Oligonucleotide from rat MASP-2 <400> 64 gaggtgacgc aggaggggca ttagtgttt 29 <210> 65 <211> 37 <212> DNA <213> Artificial Sequence <220> <223> Oligonucleotide from rat MASP-2 <400> 65 ctagaaacac taatgcccct cctgcgtcac ctctgca 37  

Claims (58)

2. A method of inhibiting MASP-2-dependent complement activation in a subject in need thereof, comprising administering to the subject an amount of a MASP-2 inhibitor effective to inhibit MASP-2-dependent complement activation. 7. The method of claim 1, wherein the MASP-2 inhibitor specifically binds to a polypeptide comprising SEQ ID NO: 6. 4. The method of claim 1, wherein the MASP-2 inhibitor specifically binds to a polypeptide comprising SEQ ID NO: 6 with an affinity that is at least ten times greater than binding to other antigens in the complement system. 3. The method of claim 2, wherein the MASP-2 inhibitor binds to the polypeptide at a position within amino acid residues 1-176 of SEQ ID NO: 6. The method of claim 1, wherein the MASP-2 inhibitor is an antibody or a fragment thereof that specifically binds to a portion of SEQ ID NO: 6. 6. The method of claim 5, wherein the antibody or fragment thereof is monoclonal. 6. The method of claim 5, wherein the antibody or fragment thereof is polyclonal. 6. The method of claim 5, wherein the antibody or a fragment thereof is a recombinant antibody. 6. The method of claim 5, wherein the antibody has reduced effector function. 6. The method of claim 5, wherein the antibody is a chimeric, human adaptive or human antibody. 6. The method of claim 5, wherein the antibody is produced in an MASP-2 deficient gene insertion animal. The method of claim 1, wherein the MASP-2 inhibitor is selected from the group consisting of human MASP-2, human MBL inhibiting MASP-2, human H-picoline inhibiting MASP-2, -2 &lt; / RTI &gt; and human C4 inhibiting MASP-2. &Lt; RTI ID = 0.0 &gt; 11. &lt; / RTI &gt; The method of claim 1, wherein the MASP-2 inhibitor is a non-peptide agent that specifically binds to a polypeptide comprising SEQ ID NO: 6. 14. The method of claim 13, wherein the MASP-2 inhibitor binds to the polypeptide at a position within amino acid residues 1-176 of SEQ ID NO: 6. Comprising administering to a subject an amount of a MASP-2 inhibitor effective to selectively inhibit MASP-2-dependent complement activation without substantially inhibiting C1q-dependent complement activation. Dependent &lt; / RTI &gt; 16. The method of claim 15, wherein the MASP-2 inhibitor specifically binds to a polypeptide comprising SEQ ID NO: 6. 17. The method of claim 16, wherein the MASP-2 inhibitor binds to the polypeptide at a position within amino acid residues 1-176 of SEQ ID NO: 6. 16. The method of claim 15, wherein the MASP-2 inhibitor is an antibody or a fragment thereof that specifically binds to a portion of SEQ ID NO: 6. 19. The method of claim 18, wherein the antibody or fragment thereof is monoclonal. 19. The method of claim 18, wherein the antibody is a chimeric, human-adapted, or human antibody. 19. The method of claim 18, wherein the antibody is produced in an MASP-2 deficient gene insertion animal. 16. The method of claim 15, wherein the MASP-2 inhibitor is human MASP-2, human MBL inhibiting MASP-2, human H-picoline inhibiting MASP-2, human L- -2. &Lt; / RTI &gt; 16. The method of claim 15, wherein the MASP-2 inhibitor is a non-peptidic agent that specifically binds to a polypeptide comprising SEQ ID NO: 6. A composition for inhibiting MASP-2-dependent complement activation, comprising a therapeutically effective amount of a MASP-2 inhibitor and a pharmaceutically acceptable carrier. 2. A method for the manufacture of a medicament for use in inhibiting the effect of MASP-2-dependent complement activation in a living subject in need thereof, comprising combining a therapeutically effective amount of a MASP-2 inhibitor in a pharmaceutical carrier. 2. A method of treating a subject suffering from MASP-2-dependent complement-mediated vascular symptoms comprising administering an amount of a MASP-2 inhibitor effective to inhibit MASP-2-dependent complement activation. 27. The method of claim 26, wherein the vascular symptom is selected from the group consisting of cardiovascular symptoms, cerebrovascular symptoms, peripheral (e.g., musculoskeletal) vascular symptoms, renal vascular symptoms, mesenteric / bowel vascular symptoms, vascular remodeling upon transplantation and / Vasculitis associated with rheumatoid arthritis, vasculitis of the immune complex, vasculitis complex, Takayama sickness, dilated cardiomyopathy, diabetic vascular disease, Kawasaki disease (arteritis), venous air embolism (VGE), and vasculitis associated with systemic lupus erythematosus Restenosis after stent insertion, rotator cuff resection and percutaneous transluminal coronary angioplasty (PTCA). 2. A method of treating a subject suffering from a MASP-2-dependent complement mediated symptom associated with ischemia-reperfusion injury comprising administering an amount of a MASP-2 inhibitor effective to inhibit MASP-2-dependent complement activation. 29. The method of claim 28, wherein the ischemia-reperfusion injury is selected from the group consisting of aortic aneurysm repair, cardiopulmonary bypass surgery, angioplasty involving organ transplantation and / or limb / fingers reattachment, stroke, myocardial infarction, and shock and / Lt; RTI ID = 0.0 &gt; a &lt; / RTI &gt; hemodynamic resuscitation. A method of treating and / or preventing atherosclerosis in a subject in need thereof, comprising administering an amount of a MASP-2 inhibitor effective to inhibit MASP-2-dependent complement activation. 2. A method of treating a subject suffering from a MASP-2-dependent complement mediated symptom associated with an inflammatory gastrointestinal disorder, comprising administering an amount of a MASP-2 inhibitor effective to inhibit MASP-2-dependent complement activation. 32. The method of claim 31, wherein the inflammatory gastrointestinal tract disorder is selected from the group consisting of pancreatitis, Crohn's disease, ulcerative colitis, irritable bowel syndrome and diverticulitis. 2. A method of treating a subject suffering from MASP-2-dependent complement-mediated pulmonary circulation, comprising the step of administering an amount of a MASP-2 inhibitor effective to inhibit MASP-2-dependent complement activation. 34. The method of claim 33 wherein the pulmonary circulation is selected from the group consisting of acute respiratory distress syndrome, transfusion-related acute lung injury, ischemia / reperfusion acute lung injury, chronic obstructive pulmonary disease, asthma, Wegener's granulomatosis, Characterized in that the method is selected from the group consisting of aspiration syndrome, closed bronchitis syndrome, idiopathic pulmonary fibrosis, secondary acute lung injury due to burn, non-cardiac pulmonary edema, transfusion-related respiratory depression and pneumonia. Dependent complement activation in a subject who has undergone, is or is undergoing an extracorporeal reperfusion procedure comprising administering an amount of a MASP-2 inhibitor effective to inhibit MASP-2-dependent complement activation. How to suppress. 36. The method of claim 35, wherein the cardiopulmonary bypass procedure is selected from the group consisting of hemodialysis, plasma exchange, leukocyte subsets, extracorporeal oxygen supply (ECMO), heparin-induced maximal oxygenated LDL deposition (HELP) ). &Lt; / RTI &gt; 2. A method of treating a subject suffering from a MASP-2-dependent complement-mediated musculoskeletal symptom, comprising administering an amount of a MASP-2 inhibitor effective to inhibit MASP-2-dependent complement activation. 37. The method of claim 37, wherein the musculoskeletal symptoms are selected from the group consisting of osteoarthritis, rheumatoid arthritis, rheumatoid arthritis, gout, neuropathic arthropathy, psoriatic arthritis, spondyloarthropathies, arthritic arthritis, muscle regressive atrophy and systemic lupus erythematosus &Lt; / RTI &gt; 2. A method of treating a subject suffering from MASP-2-dependent complement-mediated renal impairment, comprising administering an amount of a MASP-2 inhibitor effective to inhibit MASP-2-dependent complement activation. 40. The method of claim 39, wherein the kidney condition is selected from the group consisting of glomerulonephritic glomerulonephritis, glomerulonephritis, glomerular glomerulonephritis (glomerular capillary glomerulonephritis), glomerulonephritis after acute infection (glomerulonephritis after a streptococcal infection) Nephritis, Nephritis, Nephritis, Nephritis, Nephritis, Nephritis, Nephritis, Nephritis, Nephritis, Nephritis, Nephritis. 2. A method of treating a subject suffering from a MASP-2-dependent complement mediated skin condition comprising administering an amount of a MASP-2 inhibitor effective to inhibit MASP-2-dependent complement activation. 42. The method of claim 41, wherein the dermatological condition is selected from the group consisting of psoriasis, autoimmune, dermatosis, eosinophil-enhanced cotton, bullous pemphigoid, acquired papillary epidermolysis, gynecological herpes, thermal burn injury and chemical burn injury &Lt; / RTI &gt; MASP-2-dependent complement activation in a subject undergoing or undergoing organs or tissue transplant surgery, comprising administering an amount of a MASP-2 inhibitor effective to inhibit MASP-2-dependent complement activation. / RTI &gt; 44. The method of claim 43, wherein the transplant operation is selected from the group consisting of organ transplantation, organ transplantation, and organ transplantation. 2. A method of treating a subject suffering from a MASP-2-dependent complement mediated symptom associated with a neurological disorder or impairment, comprising administering an amount of a MASP-2 inhibitor effective to inhibit MASP-dependent complement activation. 45. The method of claim 45, wherein the neurological disorder or injury is selected from the group consisting of multiple sclerosis, myasthenia gravis, Huntington's chorea, amyotrophic lateral sclerosis, acute spastic neuropathic pain, post-stroke reperfusion, degenerative disk, brain trauma, Parkinson's disease, Alzheimer's disease, , Brain trauma and / or hemorrhage, dehydration, and meningitis. 2. A method of treating a subject suffering from a MASP-2-dependent complement mediated symptom associated with a blood disorder comprising administering an amount of a MASP-2 inhibitor effective to inhibit MASP-2-dependent complement activation. 48. The method of claim 47, wherein the blood disorder is selected from the group consisting of sepsis, severe sepsis, septic shock, acute respiratory distress syndrome due to sepsis, systemic inflammatory response syndrome, hemorrhagic shock, hemolytic anemia, autoimmune thrombotic hemolytic anemia, &Lt; / RTI &gt; 2. A method of treating a subject suffering from MASP-2-dependent complement mediated symptoms associated with a genitourinary condition, comprising administering an amount of a MASP-2 inhibitor effective to inhibit MASP-2-dependent complement activation. 53. The method of claim 49, wherein the symptom of genitourinary disorder is selected from the group consisting of pain bladder disease, sensory bladder disease, chronic aseptic cystitis, interstitial cystitis, infertility, placental dysfunction and lactation, . (Type 1 diabetes or insulin-dependent diabetes) and / or type 1 or type 2 diabetes, comprising injecting an amount of a MASP-2 inhibitor effective to inhibit MASP- (Adult) A method of treating a subject suffering from MASP-2-dependent complement mediated symptoms associated with complications associated with diabetes. 52. The method of claim 51, wherein the complications associated with Type 1 or Type 2 diabetes are selected from the group consisting of vascular diseases, neuropathy and retinopathy. Administering an amount of a MASP-2 inhibitor effective to inhibit MASP-2-dependent complement activation. The use of MASP-2 inhibitors in a subject who has undergone, is or is undergoing chemotherapeutic treatment and / Dependent &lt; / RTI &gt; A method of treating a subject suffering from a malignant tumor, comprising administering an amount of a MASP-2 inhibitor effective to inhibit MASP-2-dependent complement activation. A method of treating a subject suffering from an endocrine disorder, comprising administering an amount of a MASP-2 inhibitor effective to inhibit MASP-2-dependent complement activation. 56. The method of claim 55, wherein the endocrine disorder is selected from the group consisting of Hashimoto's thyroiditis, stress, anxiety, prolactin from the pituitary gland, growth or other hormone disorders associated with controlled release of insulin-like growth factor and adrenocorticotropin &Lt; / RTI &gt; Comprising administering an amount of a MASP-2 inhibitor effective to inhibit MASP-2-dependent complement activation. 57. The method of claim 57, wherein the ophthalmic condition is age-related macular degeneration.

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